US20220211737A1 - Compositions and methods for treatment of friedreichs ataxia - Google Patents

Compositions and methods for treatment of friedreichs ataxia Download PDF

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US20220211737A1
US20220211737A1 US17/434,318 US202017434318A US2022211737A1 US 20220211737 A1 US20220211737 A1 US 20220211737A1 US 202017434318 A US202017434318 A US 202017434318A US 2022211737 A1 US2022211737 A1 US 2022211737A1
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fxn
nucleic acid
utr
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Darin FALK
Edgardo Rodriguez
Madhurima SAHA
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Prevail Therapeutics Inc
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Lacerta Therapeutics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material

Definitions

  • the present disclosure generally relates to compositions and methods for treatment of Friedreich's Ataxia (FA).
  • FA Friedreich's Ataxia
  • Friedreich's ataxia is an autosomal recessive disease and is the most common form of hereditary ataxia in the United States, affecting ⁇ 1 in every 40,000 people. It is caused by expansion of a GAA triplet in the first intron of the FXN gene ( FIG. 1B ). This mutation, not present in healthy individuals ( FIG. 1A ) alters FIN transcription and decreases the expression of frataxin (FXN), a small mitochondrial protein involved in iron sulfur cluster assembly (Cook et al. Friedreich's ataxia: clinical features, pathogenesis, and management. Br Med Bull, 2017:124(1):19-30). Individuals who inherit two alleles with a trinucleotide repeat expansion in the FXN gene will likely develop FA.
  • FXN frataxin
  • FA central nervous system
  • CNS central nervous system
  • heart disease e.g. hypertrophic cardiomyopathy or myocardial fibrosis
  • CNS central nervous system
  • a less severe gene mutation accounts for disease onset alter the age of 25.
  • most affected individuals are confined to a wheelchair due to loss of sufficient motor control (Rummey et al, Predictors of Loss of Ambulation in Friedreich's Ataxia ECinicalMedicine. 2020 Jan. 8; 18:100213).
  • patients become incapacitated and generally die in early adulthood from heart disease.
  • FA central nervous system
  • nucleic acid constructs for modulating expression of human frataxin in vitro, ex vivo and in vivo.
  • the nucleic acid constructs include a nucleic acid sequence including a human frataxin 5′ untranslated region (5′UTR FXN) and a nucleic acid sequence encoding human frataxin (FXN).
  • the nucleic acid sequence encodes a human frataxin having the amino acid sequence of SEQ ID NO: 60.
  • the nucleic acid sequence encoding human FXN has at least 85% sequence identity to SEQ ID NO: 1.
  • the 5′UTR FXN has at least 85% sequence identity to SEQ ID NO: 2.
  • the 5′UTR FXN includes SEQ ID NO: 2. In some embodiments, the 5′UTR FXN includes a CCCTC-binding factor (CTCF) binding site. In certain embodiments, the CTCF binding site includes any one of SEQ ID NOs: 3 or 16-21.
  • CCCTC-binding factor CCCTC-binding factor
  • the nucleic acid sequence encoding human FXN is codon-optimized. In some embodiments, the 5′UTR FXN is located upstream of the nucleic acid sequence encoding human FXN.
  • the nucleic acid construct further includes an intron, wherein the intron is positioned downstream of the 5′UTR FXN and upstream of the nucleic acid encoding human FXN.
  • the nucleic acid construct further includes a nucleic acid sequence including an RNA polymerase II promoter.
  • the nucleic acid construct includes, in the following order:
  • RNA polymerase II promoter a nucleic acid sequence including a RNA polymerase II promoter
  • nucleic acid sequence including a 5′UTR FXN a nucleic acid sequence including a 5′UTR FXN
  • nucleic acid sequence encoding human FXN has at least 85% sequence identity to SEQ ID NO: 1, and wherein the RNA polymerase II promoter is operably linked to the 5′UTR FXN and the nucleic acid sequence encoding a human FXN.
  • the nucleic acid construct includes, in the following order: (a) a nucleic acid sequence including RNA polymerase II promoter; (b) a nucleic acid sequence including a 5′UTR FXN; (c) an intron; and (d) a nucleic acid sequence encoding human FXN, wherein the RNA polymerase II promoter is operably linked to the 5′UTR FXN and the nucleic acid sequence encoding a human FXN.
  • the nucleic acid sequence encodes a amino acid sequence comprising SEQ ID NO: 60.
  • the nucleic acid sequence encoding human FXN has at least 85% sequence identity to SEQ ID NO: 1, and
  • the RNA polymerase II promoter is selected from the group consisting of a desmin promoter, a CBA promoter and a human frataxin promoter. In some embodiments, the RNA polymerase II promoter includes SEQ ID NO: 4 or SEQ ID NO: 5.
  • the RNA polymerase II promoter is a spatially-restricted promoter.
  • the spatially-restricted promoter is selected from the group consisting of: a neuron-specific promoter, a cardiomyocyte-specific promoter, a skeletal muscle-specific promoter, a liver-specific promoter, astrocyte-specific promoter, microglial-specific promoter, and oligodendrocyte-specific promoter.
  • the nucleic acid construct further includes a pair of inverted terminal repeats (ITR), wherein the nucleic acid construct is flanked on each said by an ITR.
  • ITR inverted terminal repeats
  • the viral vector is an adeno-associated viral (AAV) vector.
  • AAV vector is selected from the group consisting of AAV1 serotype vectors, AAV2 serotype vectors.
  • the recombinant AAV vector is a single-stranded or self-complementary AAV vector.
  • the recombinant AAV vector includes a nucleic acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 6, 14-15, or 24-28. In some embodiments, the recombinant AAV vector any one of SEQ ID NOs: 6, 14-15 or 24-28.
  • nucleic acid including any of the recombinant AAV vectors described herein is a plasmid.
  • recombinant AAV particles including any of the recombinant AAV vectors provided herein.
  • a plurality of any of the AAV particles described herein is also provided.
  • composition including a plurality of any of the AAV particles provided herein.
  • the genetically modified cell can be an in vitro, ex vivo or in vivo modified cell, In some embodiments, the genetically modified cell is selected from the group consisting of a human neuron, a human cardiomyocyte, a human smooth muscle myocyte, a human skeletal myocyte, and a human hepatocyte.
  • the methods include administering to a subject having FA, a therapeutically effective amount of any of the recombinant AAV particles provided herein.
  • kits for modulating expression of FXN in a human cell include introducing into the human cell, any of the recombinant AAV vectors provided herein.
  • adenosine triphosphate (ATP) concentration in a human cell of a subject with FA include administering to the subject a therapeutically effective amount of any of the recombinant AAV particles provided herein.
  • the human cell is selected from the group consisting of: a neuron, a cardiomyocyte, a smooth muscle myocyte, a skeletal myocyte, and a hepatocyte.
  • the methods include administering a therapeutically effective amount of any of the recombinant AAV particles provided herein.
  • the human cell is selected from the group consisting of: a neuron, a cardiomyocyte, a smooth muscle myocyte, a skeletal myocyte, and a hepatocyte.
  • the present application includes the following figures.
  • the figures are intended to illustrate certain embodiments and/or features of the compositions and methods, and to supplement any description(s) of the compositions and methods.
  • the figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.
  • FIGS. 1A-1B illustrate a genomic DNA sequence and expression thereof in a healthy individual (e.g., an individual without FA) and in an individual with FA.
  • FIG. 1A illustrates a genomic DNA sequence and expression thereof in a healthy individual.
  • FIG. 1B illustrates a genomic DNA sequence and expression thereof in an individual with FA.
  • FIGS. 2A-2B illustrate exogenous nucleic acids and expression thereof in an individual with FA.
  • FIG. 2A illustrates an exogenous nucleic acid and non-modulated expression thereof in an individual with FA.
  • FIG. 2B illustrates an exogenous nucleic acid and modulated expression thereof in an individual with FA.
  • FIG. 3 illustrates an AAV vector sequence including a human FXN ORF operably linked to a 5′ITR FXN (SEQ ID NO: 2) and a RNA polymerase II promoter.
  • FIG. 4 shows a Western blot performed on whole cell extracts of human embryonic kidney (HEK) 293 cells.
  • FIG. 5 shows a Western blot performed on whole cell extracts of SK-N-SH cells
  • FIG. 6 shows a Western blot performed on whole cell extracts of C2C12 mouse myoblast cells.
  • FIG. 7 shows a bar graph showing ATP content of C2C12 mouse myoblast cells
  • FIG. 8 shows a Western blot performed on whole cell extracts of C2C12 mouse myoblast cells.
  • FIG. 9 shows a bar graph showing qPCR results of whole cells extracts of C2C12 mouse myoblast cells.
  • FIGS. 10A-10B are maps of different vectors, different promoters, and/or a codon-optimized sequence encoding human frataxin with and without a 5′ UTR.
  • FIG. 10A is a map of the LP1001 AAV vector including a desmin promoter, a 5′ UTR and a codon-optimized sequence encoding human frataxin.
  • FIG. 10B is a map of the LP1002 AAV vector including a chicken ⁇ -actin (CBA) promoter and a codon-optimized sequence encoding human frataxin.
  • CBA chicken ⁇ -actin
  • FIG. 10C is a map of the LP1003 AAV vector including a CBA promoter, a 5′ UTR and a codon-optimized sequence encoding human frataxin.
  • FIG. 1D is a map of the LP1004 AAV vector including a desmin (DES) promoter and a codon-optimized sequence encoding human frataxin.
  • FIG. 10E is a map of the LP1049 AAV vector including a CBA promoter, a 3′ UTR and a codon-optimized sequence encoding human frataxin.
  • FIG. 10F is a map of a AAV8TM vector (SEQ ID NO: 61).
  • FIGS. 11A-C show the toxicity of plasmid-induced FXN expression in control and patient fibroblasts, Fibroblasts were transfected with plasmids expressing FXN controlled by a CBA or DES promoter with or without a 5′ UTR.
  • A Fibroblasts from normal, healthy individuals or Friedreich's ataxia patients were transfected with plasmid constructs as indicated.
  • B DNA content was measured by CyQUANT Proliferation Assay to evaluate potential plasmid toxicity in fibroblast cultures. The line represents the value at which no toxicity was observed.
  • plasmids containing a 5′UTR showed ⁇ 200% reduced toxicity compared to constructs without a 5′UTR.
  • FIGS. 12A-C show human FXN levels in transfected fibroblasts.
  • A Western blot images and
  • FIGS. 13A-I show the results of a comparison between the 5′ untranslated region and the 3′ untranslated regions of frataxin plasmids in vitro.
  • Fibroblasts from healthy (control) and FA patients were transfected with 5 ⁇ g of plasmid expressing FX with or without a UTR under the control of CBA promoter (Table 2).
  • Cells that were not transfected (no plasmid) and cells transfected with CBA-GFP were used as negative and transfection control, respectively.
  • Cells were imaged in a 24-well plate for visualization of cell confluency after transfection of constructs ( FIG. 13A ).
  • Cell viability was measured after transfection by CyQUANT assay ( FIG. 13B ).
  • CBA-FXN decreased cell viability in control fibroblasts when compared to CBA-5′-FXN.
  • FA fibroblasts do not show the same distribution of toxicity ( FIG. 13C ).
  • ATP content was measured in non- and transfected cells ( FIG. 13D ).
  • Detection of frataxin overexpression by ELISA was ⁇ ⁇ 16 times higher in CBA-FXN transfected control fibroblasts above endogenous frataxin levels.
  • CBA-5′-FXN and CBA-3-FXN were ⁇ 10 times higher in expression when compared to endogenous frataxin expression (CBA-GFP).
  • FIG. 13E-3G Densitometric analysis was performed after western blot directed against frataxin and GAPDH ( FIG. 13E-3G ). Immunocytochemistry detection of frataxin and tomm20 confirmed co-localization of frataxin in mitochondria ( FIG. 13H ) and staining of control and diseased cells in under each condition was reflective of protein expression ( FIG. 13I ).
  • FIGS. 14A-B show the titration of plasmid content to reduce the toxicity observed in vitro.
  • a dose-response (ug DNA) curve was performed.
  • Control and FA fibroblasts were transfected with plasmids constructs expressing FXN driven by the CBA promoter (Table 2).
  • 1 ⁇ -1.25 ⁇ g, 2 ⁇ -25 ⁇ g and 4 ⁇ -5 ⁇ g represents the plasmid concentration of each condition.
  • Cell toxicity analysis revealed CBA-FXN to result in the highest toxicity, however, titration of the plasmid (i.e, reduction) attenuated the degree of cell death.
  • FIG. 15 shows endogenous frataxin levels in wild-type nice.
  • FIG. 16 shows levels of human frataxin in wild-type mice following intravenous administration of AAV8TM-DES-5′UTR-FXN
  • FIGS. 17A-D show human frataxin expression in wild-type mice injected intra cisterna magna (ICM) and intramuscularly (1M) with AAV8TM-DES-5′UTR-FXN (A-C doses) Detection of human frataxin in the brain, spinal cord and skeletal muscles.
  • ICM intra cisterna magna
  • 1M intramuscularly
  • AAV8TM-DES-5′UTR-FXN A-C doses
  • FIG. 18 shows the effects of intron placement on frataxin expression. Constructs that do not include a 5′UTR results in highly significant expression (lanes 3 and 6). Inclusion of the 5′ UTR between the intron and FXN results in low FNX expression (lane 5). Inclusion of the 5′UTR, an intron and FXN, in that order, results in desired FXN expression levels (lanes 2 and 4).
  • FIG. 19 shows an exemplary 5 UTR FXN sequence (SEQ ID NO: 33) with regulatory regions.
  • SEQ ID NO: 1 is an FXN nucleotide sequence which is a codon-optimized ORF from a frataxin cDNA sequence.
  • SEQ ID NO: 2 is an exemplary 5′ UTR FXN sequence.
  • SEQ ID NO: 3 is a CTCF protein binding site.
  • SEQ ID NO. 4 is a desmin promoter sequence.
  • SEQ ID NO: 5 is a chicken beta actin (CBA) promoter sequence.
  • SEQ ID NO: 6 is a recombinant AAV vector sequence including a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a desmin promoter sequence (SEQ ID NO: 4) and further includes a desmin 5′UTR (SEQ ID NO: 22) and 5′UTR FXN sequence (SEQ ID NO: 2) operably positioned between the desmin promoter sequence and the human FXN nucleotide sequence.
  • SEQ ID NO: 7 is a plasmid sequence that encodes a recombinant AAV vector.
  • the recombinant AAV vector includes a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a desmin promoter sequence (SEQ ID NO: 4) and flutter includes a desmin 5′UTR (SEQ ID NO: 22) operably positioned between the desmin promoter sequence and the human FXN nucleotide sequence.
  • SEQ ID NO: S is a plasmid sequence that encodes a recombinant AAV vector.
  • the recombinant AAV vector includes a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a desmin promoter sequence (SEQ ID NO: 4) and further includes a desmin 5′UTR (SEQ ID NO: 22) and 5′UTR FXN sequence (SEQ ID NO: 2) operably positioned between the desmin promoter sequence and the human FXN nucleotide sequence.
  • SEQ ID NO: 9 is a plasmid sequence similar to SEQ ID NO: 7 except that it further includes a C-terminal V5 epitope tag in-frame with the human FXN nucleotide sequence.
  • SEQ ID NO: 10 is a plasmid sequence similar to SEQ ID NO: S except that it further includes a. C-terminal V5 epitope tag in-frame with the human FXN nucleotide sequence
  • SEQ ID NO: 11 is a plasmid sequence including a recombinant AAV vector.
  • the recombinant AAV vector includes a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a CBA promoter sequence (SEQ ID NO: 5) and further includes a CBA 5′UTR (SEQ ID NO: 23) and 5′UTR FXN sequence (SEQ ID NO: 2) operably positioned between the CBA promoter sequence and the human FXN nucleotide sequence.
  • SEQ ID NO: 12 is a plasmid sequence including a recombinant AAV vector.
  • the recombinant AAV vector includes a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a CBA promoter sequence (SEQ ID NO: 5) and further includes a CBA 5′UTR (SEQ ID NO: 23) operably positioned between the CBA promoter sequence and the human FXN nucleotide sequence.
  • SEQ ID NO: 13 is a plasmid sequence including a recombinant AAV vector.
  • the recombinant AAV vector includes a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a desmin promoter sequence (SEQ ID NO: 4) and further includes a 5′UTR FXN sequence (SEQ ID NO: 2) operably positioned between the desmin promoter sequence and the human FXN nucleotide sequence.
  • SEQ ID NO: 14 is a recombinant AAV vector sequence including a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a CBA promoter sequence (SEQ ID NO: 5) and further includes a CBA 5′UTR (SEQ ID NO: 23) and 5′UTR FXN sequence (SEQ ID NO: 2) operably positioned between the CBA promoter sequence and the human FXN nucleotide sequence.
  • SEQ ID NO: 1 codon-optimized human FXN nucleotide sequence
  • SEQ ID NO: 5′UTR SEQ ID NO: 23
  • 5′UTR FXN sequence SEQ ID NO: 2
  • SEQ ID NO: 15 is a recombinant AAV vector sequence including a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a desmin promoter sequence (SEQ ID NO: 4) and further includes a 5′UTR FXN sequence (SEQ ID NO: 2) operably positioned between the desmin promoter sequence and the human FXN nucleotide sequence.
  • SEQ ID Nos: 16-21 are CTCF protein binding sites.
  • SEQ ID NO: 22 is a desmin 5′UTR.
  • SEQ ID NO: 23 is a CBA 5′UTR.
  • SEQ ID NO: 24 is a plasmid sequence including a recombinant AAV vector (LP1001).
  • the recombinant AAV vector includes, in the following order, in operable linkage, a desmin promoter sequence (SEQ ID N: 4), a 5′UTR FXN sequence (SEQ ID NO: 2), an intron, a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1).
  • SEQ ID NO: 25 is a plasmid sequence including a recombinant AAV vector (LP1002).
  • the recombinant AAV vector includes, in the following order, in operable linkage, a CMV promoter sequence (SEQ ID NO: 34), a CBA promoter, (SEQ ID NO: 5, an intron and a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1).
  • SEQ ID NO: 26 is a plasmid sequence including a recombinant A-AV vector (LP1003).
  • the recombinant AAV vector includes, in the following order, in operable linkage, a CMV promoter sequence (SEQ ID NO: 34), a CBA promoter. (SEQ ID NO: 5, a 5′UTR FXN sequence (SEQ ID NO: 2), an intron, and a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1).
  • SEQ ID NO: 27 is a plasmid sequence including a recombinant AAV vector (LP1004).
  • the recombinant AAV vector includes, in the following order, in operable linkage, a desmin promoter sequence (SEQ ID NO: 4) an intron and a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1).
  • SEQ ID NO: 28 is a plasmid sequence including a recombinant A-AV vector (LP1049).
  • the recombinant AAV vector includes, in the following order, in operable linkage, a CMV promoter sequence (SEQ ID NO: 34), a CBA promoter sequence (SEQ ID NO: 5) an intron, a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) and a 3′ UTR FXN (SEQ ID NO: 35).
  • SEQ ID NO: 29 is a self-complementary plasmid sequence including AAV-CBA-EGFP (GenBauk: Accession No. MK225672).
  • SEQ ID NO: 30 is a primer.
  • SEQ ID NO: 31 is a primer.
  • SEQ ID NO: 32 is a primer.
  • SEQ ID NO: 33 is an exemplary 5′ TR FXN sequence with TFAP2 (SEQ ID NO: 57), SRF1 (SEQ ID NO: 56) and SP1 (SEQ ID NO: 58) regulatory regions.
  • SEQ ID NO: 34 is a CMV enhancer sequence.
  • SEQ ID NO: 35 is 3′ UTR FXN
  • SEQ ID NO: 36 is an intron sequence included in SEQ ID NO: 24.
  • SEQ ID NO: 37 is a modified SV40 intron with splice donor and acceptor sites.
  • SEQ ID NO: 38 is an exemplary mutated ITR sequence.
  • SEQ ID NO: 39 is an exemplary ITR sequence.
  • SEQ ID NO: 40 is a human 3′ UTR FXN.
  • SEQ ID NO: 41 is a truncated 3′UTR FXN.
  • SEQ ID NO: 42 is an exemplary frataxin promoter sequence.
  • SEQ ID NO: 43 is an exemplary frataxin promoter sequence.
  • SEQ ID NO: 44 is an ampicillin resistance gene.
  • SEQ ID NO: 45 is a kanamycin resistance gene.
  • SEQ ID NO: 46 is an exemplary 3′ UTR FXN sequence that does not include a putative iron binding domain.
  • SEQ ID NO: 47 is an exemplary 3′ UTR FXN sequence that does not include a mitochondrial localization signal.
  • SEQ ID NO: 48 is an exemplary 5′ UTR FXN sequence that does not include a L2 retrotransposable element (SEQ ID NO: 54).
  • SEQ ID NO: 49 is an exemplary 5′ UTR FXN sequence that does not include an alternate RNA export signal (SEQ ID NO: 55)
  • SEQ ID NO: 50 is an exemplary 5′ UTR FXN sequence that does not include a CTCF domain.
  • SEQ ID NO: 51 is an exemplary 5′ UTR FXN sequence.
  • SEQ ID NO: 52 is an exemplary 5′ UTR FXN sequence.
  • SEQ ID NO: 53 is an exemplary 5′ UTR FXN sequence that does not include the catalytic binding domain (SEQ ID NO: 59).
  • SEQ ID NO: 54 is an L2 retrotransposable element
  • SEQ ID NO: 55 is an alternate RNA export signal.
  • SEQ ID NO: 56 is an SRF regulatory sequence.
  • SEQ ID NO: 57 is a TFAP2 regulatory sequence.
  • SEQ ID NO: 58 is a regulatory SP1 sequence.
  • SEQ ID NO. 59 is an aconitase binding domain.
  • SEQ ID NO. 60 is an exemplary amino acid sequence for human frataxin.
  • SEQ ID NO: 61 is an AA8V triple-capsid mutant vector sequence.
  • SEQ ID NO: 62 is an exemplary 5′ UTR FXN.
  • SEQ ID NO: 63 is a nucleic acid sequence encoding a bovine growth hormone polyaclenlyation sequence.
  • compositions for use in methods of treating FA in a subject include, but are not limited to, novel nucleic acid constructs, recombinant viral vectors and cells including a human frataxin 5′UTR (5′UTR FXN) and a nucleic acid sequence encoding FXN.
  • nucleic acid constructs including a human frataxin 5′ untranslated region (5′UTR FXN) and a nucleic acid sequence encoding human frataxin (FXN).
  • the nucleic acid sequence encoding human FXN has at least 85% sequence identity to SEQ ID NO: 1 (as set forth below).
  • the nucleic acid sequence encoding human FXN is not a naturally-occurring nucleotide sequence encoding human FXN.
  • a 5′UTR FXN is an untranslated nucleic acid sequence, that is upstream from the initiation codon of a nucleic acid encoding human FXN.
  • a 5′ UTR FXN can modulate FXN expression. Modulated FN expression may be desired to achieve modulated physiological levels of FXN expression and mitochondrial respiration and thereby avoid non-modulated, elevated physiological levels of FX expression and reduced mitochondrial respiration.
  • this modulation can be achieved via cis effects of the 5′ UTR FXN on transcription and/or translation of mRNA transcribed from a nucleic acid of the present disclosure encoding the 5′ UTR and the human FXN nucleotide sequence.
  • the regulatory elements within the 5′ UTR FXN are shown in FIG. 19 (SEQ ID NO: 33).
  • the 5′UTR FXN can include a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2 (as set forth below) or a fragment thereof.
  • the 5′UTR FXN can include a nucleotide sequence including SEQ ID NO: 2 or a fragment thereof.
  • the 5′UTR FXN can include SEQ ID NO: SEQ ID NO: 33, SEQ ID NO: 48. SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 62.
  • the fragment can be at least about 2, 3, 4, 5, 6, 7, &, 9, 10, 20, 30, 40, 50 or more nucleotides shorter, at either or both ends of SEQ ID NO: 2.
  • the nucleic acid sequence including the 5′ UTR FXN is not a full-length FXN promoter.
  • the nucleotide sequence including the 5′ UTR FXN does not include, SEQ ID NO: 42 or SEQ ID NO: 43.
  • the nucleic acid sequence including the 5′ UTR FXN is a nucleic acid sequence that includes SEQ ID NO: 2, SEQ ID NO: 33, SEQ ID NO: 48, SEQ ID NO: 49.
  • the nucleic acid sequence including the 5′ UTR FXN includes SEQ ID NO: 2 and is not a nucleic acid sequence that is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 base pairs shorter in length, on either end, than SEQ ID NO: 42.
  • the 5′UTR FXN can be located upstream of the nucleic acid sequence encoding human FXN for example, a nucleic acid sequence encoding SEQ ID NO: 60.
  • the 5′UTR FXN can include a CTCF binding site.
  • the CTCF binding site can include a nucleotide sequence including at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least one of SEQ ID NOs: 3 or 16-21.
  • the CTCF binding site can include at least one of SEQ ID NOs: 3 or 16-21.
  • the 5′UTR FXN can include at least one CTCF binding site including any one of SEQ ID NOs: 3 and 16-21.
  • the 5′ UTR FXN does not include a functional CTCF binding site, e.g., the CTCF binding site is mutated or removed from the 5′ UTR FXN.
  • An exemplary 5′UTR FXN that does not include a functional CTCF binding site is set forth herein as SEQ ID NO: 50.
  • modulated physiological levels of FXN expression refers to levels of FXN expression at the protein level which are similar to those observed in wild-type cells.
  • modulated physiological levels of FXN expression in muscle- or nerve-derived cells including homozygous GAA repeat expansion FXN alleles treated according to methods of the present disclosure can display FXN expression levels similar to wild-type cells of a similar or isogenic background.
  • Such “modulated physiological levels of FXN expression” can reduce negative effects on cellular mitochondria function in diseased cells due to a lack of sufficient FXN or due to a harmful excess of FXN, such as an excess due to non-modulated expression of the FXN gene.
  • Modulated physiological levels of FXN expression” at the protein level can be similar to that of wild-type cells.
  • the modulated physiological levels of FXN expression at the protein level can be at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%1 ⁇ 2 at least 195%, or at least 200% of the FXN protein level in
  • including a 5′UTR FXN in a nucleic construct results in a level of FXN expression in a cell that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90% lower than the level of FXN expression in a cell with a nucleic acid construct that does not include a 5′ UTR FXN (e.g., a construct including a promoter and a nucleic acid encoding human FXN).
  • a nucleic acid sequence encoding human FXN or a human FX nucleotide sequence
  • a human frataxin FXN cDNA sequence e.g., SEQ ID NO: 1
  • Any nucleic acid sequence encoding human FXN can be operably linked to a 5′ UTR FXN described herein, including naturally and non-naturally occurring nucleic acids that encode human FXN.
  • the nucleic acid sequence encodes SEQ ID NO: 60, or SEQ ID NO: 60 with one more conservative substitutions.
  • SEQ ID NO: 1 is an exemplary codon-optimized nucleic acid sequence that encodes human frataxin protein (SEQ ID NO: 60).
  • An exemplary amino acid sequence for human frataxin can also be found under GenBank Accession No. NP_000135.2.
  • the FXN nucleotide sequence can have at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.
  • the FXN nucleotide sequence can include SEQ ID NO: 1.
  • the FXN nucleotide sequence is codon-optimized for expression in the cell to be infected by any of the recombinant AAV vectors or particles described herein.
  • the recombinant AAV infected cell is a human cell
  • a human codon-optimized polynucleotide encoding FXN for example, SEQ ID NO: 1
  • Methods for codon-optimization are known in the art. See, for example, Inouye et al. “Codon optimization of genes for efficient protein expression in mammalian cells by selection of only preferred human codons,” Protein Expression and Purification 109: 47-54 (2015)).
  • GeneOptimizer® software (Thermo Fisher Scientific, Waltham, Mass.) can also be used.
  • nucleic acid construct including a promoter operably linked to a nucleic acid sequence described herein.
  • the components or elements of the constructs described herein are operably linked to make a non-naturally occurring construct. In other words, the elements are not linked as they would be linked in the genome of naturally occurring cell.
  • a promoter is a region or a sequence located upstream and/or downstream from the start of transcription that is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • the promoter is an RNA polymerase II promoter, for example, and not to be limiting, an RNA polymerase II CORE promoter.
  • an RNA polymerase II CORE promoter is the minimal sequence that allows the basal transcription apparatus to assemble. For example, this sequence can be at 40 base pairs in length and can include a TATA box, an initiator element (Inr) and/or a downstream promoter element (DPE).
  • the promoter is an inducible promoter, for example, the promoter can be chemically or physically regulated.
  • a chemically regulated promoter and/or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal. Examples include, the tetracycline inducible promoter or a glucocorticoid inducible promoter.
  • the nucleic acids of the present invention can also be under the control of a tissue-specific promoter to promote expression of the nucleic acid in specific cells, tissues or organs.
  • a tissue-specific promoter to promote expression of the nucleic acid in specific cells, tissues or organs.
  • Any regulatable promoter such as a metallothionein promoter, a heat-shock promoter, and other regulatable promoters, of which many examples are well known in the art are also contemplated.
  • a Cre-1oxP inducible system can also be used, as well as a Flp recombinase inducible promoter system, both of which are known in the art.
  • operably linked means that a first nucleic acid sequence (e.g. a coding sequence for a protein or a non-coding RNA sequence) is covalently connected to at least a second nucleic acid sequence such that at least one of the two sequences can exert an effect on the other nucleic acid sequence.
  • a human FXN nucleotide sequence can be operably linked to a promoter sequence such that the promoter sequence can direct transcription of the human FXN nucleotide sequence, thereby contributing to expression of the human FXN nucleotide sequence.
  • a 5′ UTR FXN sequence can be operably positioned between the promoter sequence and the human FXN nucleotide sequence, such that the 5′ UTR FXN sequence can modulate expression of the human FXN nucleotide sequence.
  • the nucleic acid construct further includes a nucleic acid sequence including an RNA polymerase II promoter that is operably linked to a 5′ UTR FXN and the nucleic acid sequence encoding a human FXN.
  • the RNA polymerase II promoter can be, for example, a desmin promoter sequence (SEQ ID NO: 4), a CBA promoter sequence (SEQ ID NO: 5) or a frataxin promoter sequence, for example, SEQ ID NO: 42, SEQ ID NO: 43, or a fragment thereof.
  • the RNA polymerase II promoter can include SEQ ID NO: 4.
  • the RNA polymerase II promoter can include SEQ ID NO: 5.
  • the RNA polymerase HI promoter can include SEQ ID NO: 42 or SEQ ID NO: 43.
  • the RNA polymerase II promoter can include at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4.
  • the RNA polymerase II promoter can include at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5.
  • the RNA polymerase II promoter can include at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42 or SEQ ID NO: 43.
  • the RNA polymerase II promoter operably linked to a 5′′ UTR FXN is not the endogenous promoter that is associated with the 5′ UTR FXN, i.e., it is derived from a different protein, for example, a desmin or a CBA promoter.
  • the RNA polymerase II promoter that is operably linked to a 5′ UTR FXN and the nucleic acid sequence encoding a human FXN is not a frataxin promoter,
  • the promoter operably linked to the 5′ UTR FXN does not include SEQ ID NO: 42. SEQ ID NO: 43, or the complement of either sequence.
  • any of the construct described herein does not include a human frataxin promoter (e.g., SEQ ID NO: 42 or SEQ ID NO: 43) or a 3′ UTR FXN (e.g., SEQ ID NO: 40 or SEQ ID NO: 41).
  • a human frataxin promoter e.g., SEQ ID NO: 42 or SEQ ID NO: 43
  • a 3′ UTR FXN e.g., SEQ ID NO: 40 or SEQ ID NO: 41.
  • fragments of the desmin, CBA or a frataxin promoter can also be used in the constructs described herein, as long as the fragment retains at least 75%, 80% 85%, 90%, 95%, 100% or more of at least one activity of the promoter from which the fragment was derived, for example, the promotion of transcription of a nucleic acid in a cell (e.g., a neuronal or muscle cell.
  • the fragment can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500 or more nucleotides shorter than a wild-type promoter or a promoter sequence having at least 85% identity to a wild-type promoter sequence.
  • fragments that are at least 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 base pairs shorter in length than SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 42 or SEQ ID NO: 43 can be used as a promoter.
  • a 5′UTR of a RNA polymerase II promoter can be removed from any of the promoters or constructs of the present disclosure to further modulate expression of the human FXN nucleotide sequence.
  • SEQ ID NO: 4 and SEQ ID NO: 5 are examples of a desmin promoter and a CBA promoter respectively, that do not include a 5′ UTR.
  • an enhancer sequence for example a CMV enhancer (e.g. SEQ ID NO: 34) is operably linked to the promoter.
  • a CMV enhancer is operably linked to a promoter, for example, a CBA promoter, and an intron
  • the promoter is referred to as a CAG promoter.
  • Exemplary constructs including a CMV enhancer, a CBA promoter and an intron are provided as SEQ ID NO: 25 and SEQ ID NO: 26.
  • the RNA polymerase II promoter is a spatially-restricted promoter, for example, a tissue- or cell-specific promoter.
  • the spatially-restricted promoter can be any suitable promotor, such as those selected from the group consisting of a neuron-specific promoter, a cardiomyocyte-specific promoter, a skeletal muscle-specific promoter, a liver-specific promoter, astrocyte-specific promoter, microglial-specific promoter, and oligodendrocyte-specific promoter.
  • specific expression does not mean that the expression product is expressed only in a specific tissue(s) or cell type(s), but refers to expression substantially limited to specific tissue(s) or cell types(s).
  • the RNA polymerase II promoter can be operably linked to a 5′UTR of the RNA polymerase II promoter, the 5′UTR FXN and the FXN nucleotide sequence in the following exemplary order: RNA polymerase II promoter—5′UTR of the RNA polymerase II promoter—5′UTR FXN—nucleotide sequence.
  • the nucleic acid construct can include a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a desmin promoter sequence (SEQ ID NO: 4) and further include a desmin 5′UTR (SEQ ID NO: 22) and 5′UTR FXN sequence (SEQ ID NCO: 2) operably positioned between the desmin promoter sequence and the human FXN nucleotide sequence.
  • SEQ ID NO: 1 operably linked to a desmin promoter sequence
  • SEQ ID NO: 22 desmin 5′UTR
  • SEQ ID NCO: 2 5′UTR FXN sequence
  • the nucleic acid construct can also include a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a CBA promoter sequence (SEQ ID NO: 5) and further include a CBA 5′UTR (SEQ ID NO: 23) and 5′UTR FXN sequence (SEQ ID NO: 2) operably positioned between the CBA promoter sequence and the human FXN nucleotide sequence.
  • SEQ ID NO: 1 codon-optimized human FXN nucleotide sequence
  • SEQ ID NO: 5′UTR SEQ ID NO: 23
  • 5′UTR FXN sequence SEQ ID NO: 2
  • the RNA polymerase II promoter can be operably linked to the 5′UTR FXN and the FXN nucleotide sequence in the following exemplary order: RNA polymerase II promoter—5′UTR FXN—nucleotide sequence.
  • the nucleic acid construct can include a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a desmin promoter sequence (SEQ ID NO: 4) and further include a 5′UTR FXN sequence (SEQ ID NO: 2) operably positioned between the desmin promoter sequence and the human FXN nucleotide sequence.
  • the nucleic acid construct can also include a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a CBA promoter sequence (SEQ ID NO: 5) and further include a 5′UTR FXN sequence (SEQ ID NO: 2) operably positioned between the CBA promoter sequence and the human FXN nucleotide sequence.
  • SEQ ID NO: 1 codon-optimized human FXN nucleotide sequence
  • SEQ ID NO: 5′UTR FXN sequence SEQ ID NO: 2
  • any of the nucleic acid construct described herein can further include one or more intron nucleotide sequences.
  • the intron can be located in any suitable location within the nucleic acid construct to modulate expression.
  • the intron sequence can be located upstream of the 5′UTR FXN.
  • the intron can be located downstream of the 5′UTR FXN.
  • the intron is positioned between the 5′UTR FXN and the nucleic acid sequence encoding human FXN.
  • the intron, and splicing thereof can contribute to expression of the human FXN nucleotide sequence.
  • SEQ ID NO: 36 and SEQ ID NO: 37 are exemplary intron sequences that can be used in any of the constructs provided herein.
  • the intron is an intron that is not found in a naturally occurring nucleic acid encoding human frataxin.
  • nucleic acid construct including, in the following exemplary order: (a) a nucleic acid sequence including RNA polymerase II promoter; (b) a nucleic acid sequence including a 5′UTR FX; (c) an intron; and (d) a nucleic acid sequence encoding human FXN, wherein the RNA polymerase II promoter is operably linked to the 5′UTR FXN and the nucleic acid sequence encoding a human FXN.
  • the nucleic acid sequence encoding, human FC has at least 85% sequence identity to SEQ ID NO: 1.
  • the nucleic acid construct further includes a human frataxin 3′UTR (3′ UTR FXN) or a truncated 3′ UTR FXN positioned downstream of the coding sequence of human FXN.
  • the nucleic acid construct does not include a human frataxin 3′UTR (3′ _TR FXN) or a truncated 3′ UTR FXN, because the 3′ UTR FX or truncated 3′ UTR FXN does not include regulatory elements to modulate expression of FXN.
  • 3′ UTRs include, but are not limited to SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO 46, SEQ ID NO: 47, or a fragment thereof.
  • the nucleic acid construct further includes a pair of inverted terminal repeats (ITR), wherein the nucleic acid construct is flanked on each said by an ITR.
  • ITR sequences include, but are not limited to SEQ ID NO: 38, SEQ ID NO: 39 and their reverse complements.
  • the nucleic acid construct further includes a nucleic acid sequence encoding a polyadenylation (polyA) sequence, for example, a polyA bovine growth hormone sequence.
  • polyA polyadenylation
  • SEQ ID NO: 63 is an exemplary sequence encoding a bovine growth hormone polyA sequence.
  • nucleic acid or “nucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form, Sequences complementary to any of the sequences provided herein are also provided. It is understood that when an RNA is described, its corresponding cDNA is also described, wherein uridine is represented as thymidine. When a cDNA is described, its corresponding mRNA is also described. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • a nucleic acid sequence can include combinations of deoxyribonucleic acids and ribonucleic acids. Such deoxyribonucleic acids and ribonucleic acids include both naturally occurring molecules and synthetic analogues.
  • the polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stein-and-loop structures, and the like.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • nucleic acid sequences including, consisting of or consisting essentially of a nucleic acid sequence having at least 60% identity to any one of SEQ ID NOs. 1-63.
  • identity or “substantial identity,” as used in the context of a polynucleotide or polypeptide sequence described herein, refers to a sequence that has at least 60% sequence identity to a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%.
  • Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below.
  • BLAST BLAST using standard parameters
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.
  • These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T. and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat. Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sun probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sun probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10, and most preferably less than about 10 ⁇ 20 .
  • the recombinant nucleic acids provided herein can be included in expression cassettes for expression in a host cell or an organism of interest.
  • the cassette may additionally contain at least one additional gene or genetic element to be cotransformed into the organism. Where additional genes or elements are included, the components are operably linked.
  • the promoters of the Invention are capable of directing or driving expression of a coding sequence in a host cell.
  • Other regulatory regions i.e., transcriptional regulatory regions, and translational termination regions
  • Additional regulatory signals include, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) (hereinafter “Sambrook 11”); Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y., and the references cited therein.
  • the expression cassette can also include a selectable marker gene for the selection of transformed cells.
  • Marker genes include genes conferring antibiotic resistance, such as those conferring hygromycin resistance, kanamycin resistance, ampicillin resistance, gentamicin resistance, neomycin resistance, to name a few. Additional selectable markers are known and any can be used. Exemplary sequences for genes conferring ampicillin resistance and kanamycin resistance are provided herein as SEQ ID NO: 44 and SEQ ID NO: 45, respectively.
  • the ampicillin resistance gene in any of the constructs described herein, for example, in pLP1001, pLP1002, pLP1003, pLp1004 or pLP1049, can be replaced with a kanamycin resistance gene.
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • vectors including any of the nucleic acid constructs described herein.
  • the vector is a plasmid.
  • the vector is a recombinant viral vector.
  • the vector is a DNA vector or PNA vector.
  • viral vectors include, but are not limited to an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, a herpes simplex viral vector, or an adenoviral vector. It is understood that any of the viral vectors described herein can be packaged into viral particles or virions for administration to the subject.
  • AAV adeno-associated virus
  • the recombinant viral vector is an AV vector.
  • the viral vector is an AAV vector including a 5′ inverted terminal repeat and a 3′ inverted terminal repeat.
  • the AAV vector can be a single-stranded AAV vector or a self-complementary AAV vector.
  • a “recombinant AAV vector” refers to an AAV vector including a nucleic acid sequence that is not normally present in AAV (i.e., a polynucleotide heterologous to AAV), for example, any of the nucleic acid constructs described herein, that expresses human frataxin.
  • the heterologous nucleic acid is flanked by at least one, and generally by two, AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • the term recombinant AAV vector encompasses both rAAV vector particles and recombinant AAV vector plasmids.
  • a recombinant AAV vector may either be single-stranded (ssAAV) or self-complementary (scAAV).
  • the recombinant AAV vector includes a nucleic acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 6-14, and 24-28. In some embodiments, the recombinant AAV vector includes any one of SEQ ID NOs: 6-14, and 24-28.
  • the recombinant AAV vector can further include viral sequences for packaging. Any missing viral functions can be supplied in trans by a packaging cell.
  • recombinant AAV vectors used in gene therapy may only possess inverted terminal repeat (ITR) sequences from the recombinant AAV genome and the balance of the vector can include sequences of interest (e.g., a 5′UTR FXN and a FXN nucleotide sequence).
  • ITR sequences can be included for packaging into AAV capsids.
  • the packaging cell can also contain a plasmid that encodes other AAV genes (e.g., rep and cap), but lacks ITR sequences.
  • the plasmid that encodes rep and cap genes may not be packaged in significant amounts due to a lack of ITR sequences.
  • the packaging cell can also be infected with adenovirus as a helper virus, which can promote replication of the AAV vector and expression of A-AV genes from the plasmid that encodes rep and cap genes.
  • the packaging cell can be transfected with a helper plasmid encoding gene products of helper viruses, such as adenovirus, which promotes replication of the AAV vector and expression of AAV genes from the plasmid that encodes rep and cap genes.
  • Purification of AAV particles from a packaging cell can involve growth of the packaging cells that produces the viral vectors, followed by collection of the viral vector particles from the cell supernatant and/or from the crude lysate.
  • A-AV can then be purified, such as by ion exchange chromatography (e.g., U.S. Pat. Nos. 7,419,817 and 6,989,264), ion exchange chromatography and CsCl or iodixanol density centrifugation (e.g., PCT publication WO2011094198A10), immunoaffinity chromatography (e.g., WO2016128408) or purification using AVB Sepharose (e.g., GE Healthcare Life Sciences).
  • ion exchange chromatography e.g., U.S. Pat. Nos. 7,419,817 and 6,989,264
  • CsCl or iodixanol density centrifugation e.g., PCT publication WO2011094198
  • a recombinant AAV particle or virion is a viral particle including at least one AAV capsid protein and an encapsidated recombinant AAV vector.
  • a recombinant AAV particle is a viral particle including at least one AAV capsid protein and an encapsidated recombinant AAV vector.
  • An “.AAV virus,” AAV virion.” “AAV viral particle,” or “recombinant AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide recombinant AAV vector. If the particle includes a heterologous nucleic acid sequence (i.e.
  • a nucleic acid sequence other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell it can be referred to as a recombinant AAV vector.
  • production of recombinant AAV particles or virion necessarily includes production of a recombinant AAV vector, as such a vector is contained within a recombinant AAV particle.
  • Methods for producing AAV vectors and virions are known in the art. See, for example, Shin et al. “Recombinant Adeno-Associated Viral Vector Production and Purification,” Methods Mol. Biol. 798: 267-284 (2012)).
  • the host cell can be an in vitro, ex vivo, or in vivo host cell. Populations of any of the host cells described herein are also provided. A cell culture including one or more host cells described herein is also provided. Methods for the culture and production of many cells, including cells of bacterial (for example E. coli and other bacterial strains), animal (especially mammalian), and archaebacterial origin are available in the art. See e.g., Sambrook.
  • the host cell can be a prokaryotic cell, including, for example, a bacterial cell.
  • the cell can be a eukaryotic cell, for example, a mammalian cell.
  • the cell can be an HEK293T cell, a Chinese hamster ovary (CHO) cell, a COS-7 cell, a HELA cell, an avian cell, a myeloma cell, a Pichia cell, an insect cell or a plant cell.
  • CHO Chinese hamster ovary
  • HELA cell avian cell
  • myeloma cell a Pichia cell
  • insect cell or a plant cell A number of other suitable host cell lines have been developed and include myeloma cell lines, fibroblast cell lines, and a variety of tumor cell lines such as melanoma cell lines.
  • the vectors containing the nucleic acid segments of interest can be transferred or introduced into the host cell by well-known methods, which vary depending on the type of
  • introducing in the context of introducing a nucleic acid into a cell refers to the translocation of the nucleic acid sequence from outside a cell to inside the cell. In some cases, introducing refers to translocation of the nucleic acid from outside the cell to inside the nucleus of the cell.
  • Various methods of such translocation are contemplated, including but not limited to, electroporation, nanoparticle delivery, viral delivery, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation.
  • a targeted nuclease system e.g., an RNA-guided nuclease (for example, a CRISPR/Cas9 system), a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a megaTAL (MT) (Li et al. Signal Transduction and Targeted Therapy 5. Article No. 1 (2020)) can also be used to introduce a nucleic acid into a host cell.
  • RNA-guided nuclease for example, a CRISPR/Cas9 system
  • TALEN transcription activator-like effector nuclease
  • ZFN zinc finger nuclease
  • MT megaTAL
  • Recombinant AAV particles including the recombinant AAV vectors provided herein can include or be derived from any natural or recombinant AAV serotype.
  • the AAV particles can be, or can be based on, a serotype selected from any of the following serotypes, and variants thereof including, but not limited to, AAV1, AAV10, AAV106.1/hu.37, AAV11, AAV114.3/hu.40, AAV12, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53.
  • AAV3-3 AAV33.12/hu.17, AAV33.4hu.15, AAV33.8/hu.16, AAV3-9/rh.52, AAV3a, AAV3b, AAV4, AAV4-19/rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-lb AAV42-2, AAV42-3a.
  • AAVhEr31 AAVhu.1, AAVhu.10, AAVhu.11, AAVhu.11, AAVhu.12.
  • AAV true type AAV
  • UPENN AAV UPENN AAV 10.
  • the AAV serotype can be, or have, a mutation in the AAV9 sequence, as described by N Pulichla et al. ( Molecular Therapy 19(6):1070-1078 (2011), such as, but not limited to, AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84.
  • the AAV serotype can be, or have, a sequence as described in United States Pat. No. US6156303, such as, but not limited to, AAV3B (SEQ ID NO: 1 and 10 of U.S. Pat. No. 6,156,303).
  • AAV6 SEQ ID NO: 2, 7 and 11 of U.S. Pat. No. 6,156,303
  • AAV2 SEQ ID NO: 3 and 8 of U.S. Pat. No. 6,156,303
  • AAV3A SEQ ID NO: 4 and 9, of US6156303
  • the serotype can be AAVDJ or a variant thereof such as AAVDJ8 (or AAV-DJ8), as described by Grinmm et al. ( Journal of Virology 82(12): 5887-5911 (2008)).
  • the amino acid sequence of AAVDJ8 can include two or more mutations in order to remove the heparin binding domain (HBD).
  • HBD heparin binding domain
  • 7,588,772 can include two mutations: (1) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q Gin) and (2) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).
  • the amino acid sequence of AAVDJ8 can include three mutations: (1) K406R where lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg), (2) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q: Gin) and (3) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).
  • the AAV serotype can be, or have, a sequence as described in International Publication No. WO2015121501, such as, but not limited to, true type AAV (ttAAV) (SEQ ID NO: 2 of WO2015121501).
  • ttAAV true type AAV
  • UPenn AAV10 SEQ ID NO: 8 of WO2015121501
  • Japanese AAV10 SEQ ID NO: 9 of WO2015121501
  • AAV capsid serotype selection or use can be from a variety of species.
  • the AAV can be an avian AAV (AAAV).
  • the AAAV serotype can be, or have, a sequence as described in U.S. Pat. No. 9,238,800, such as, but not limited to, AAAV (SEQ ID NO: 1, 2, 4, 6, 8& 10, 12, and 14 of US9238800), or variants thereof.
  • the AAV can be a bovine AAV (BAAV).
  • BAAV serotype can be, or have, a sequence as described in U.S. Pat. No. 9,193,769, such as, but not limited to, BAAV (SEQ ID NO: 1 and 6 of U.S. Pat. No. 9,193,769), or variants thereof.
  • BAAV serotype can be, or have, a sequence as described in U.S. Pat. No. 7,427,396, such as, but not limited to, BAAV (SEQ ID NO: 5 and 6 of U.S. Pat. No. 7,427,396), or variants thereof.
  • the AAV can be a caprine AAV.
  • the caprine AAV serotype can be, or have, a sequence as described in U.S. Pat. No. 7,427,396, such as, but not limited to, caprine AAV (SEQ ID NO: 3 of US7427396), or variants thereof.
  • the AAV can be engineered as a hybrid AAV from two or more parental serotypes.
  • the AAV can be AAV2G9 which includes sequences from AAV2 and AAV9.
  • the AAV2G9 AAV serotype can be, or have, a sequence as described in U.S. Patent Publication No. US20160017005.
  • the AAV can be a serotype generated by the AAV9 capsid library with mutations in amino acids 390-627 (VP1 numbering) as described by Pulichla et al. (Molecular Therapy 19(6):1070-1078 (2011).
  • the serotype and corresponding nucleotide and amino acid substitutions can be, but is not limited to, AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and 1479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G. A1314T, A1642G, C1760T; Q412R.
  • AAV9.6 T1231A; F411I
  • AAV9.9 G1203A, G1785T; W595C
  • AAV9.10 A1500G. T1676C; M559T
  • AAV9.11 A1425T, A1702C, A1769T; T568P, Q590L
  • AAV9.13 A1369C, A1720T; N457H. T574S
  • AAV9.14 T1340A, T1362C, T1560C.
  • AAV9.16 A1775T; Q592L
  • AAV9.24 T1507C, T1521G; W503R
  • AAV9.26 A1337G, A1769C; Y446C, Q590P
  • AAV9.33 A1667C; D556A
  • AAV9.34 A1534G, C1794T; N512D
  • AAV9.35 A1289T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N, N98K, V606I
  • AAV9.40 A1694T, E565V
  • AAV9.41 A1348T.
  • T1362C; T450S AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V), 947 (G1241A, G1358A, A1669G, C1745T; S414N, G453D.
  • AAV9.48 C1445T, A1736T; P482L, Q579L
  • AAV9.50 A1638T, C1683T, T1805A; Q546H, L602H
  • AAV9.53 G1301A, A1405C, C1664T, G1811T; R134Q.
  • AAV9.54 C1531A; T1609A; L511 I, L537M
  • AAV9.55 T1605A; F535L
  • AAV9.58 C1475T, C1579A; T492I, H527N, AAV.59 (T1336C; Y446H)
  • AAV9.61 A1493T; N498I
  • AAV9.64 C1531A, A1617T; L5111
  • AAV9.65 C1335T.
  • the AAV can be a serotype including at least one AAV capsid CD8+ T-cell epitope.
  • the serotype can be AAV1, AAV2 or AAV8.
  • the AAV can be a variant, such as PHP.A or PHP.B as described in Deverman. 2016 . Nature Biotechnology. 34(2): 204-209.
  • the present disclosure also provides a method of generating a packaging cell that includes creating a cell line that stably expresses all of the necessary components for AAV particle production.
  • a plasmid or multiple plasmids including a recombinant AAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the recombinant AAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell.
  • AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl, Acad. S6.
  • the packaging cell line can then be infected with a helper virus, such as adenovirus.
  • helper virus such as adenovirus.
  • AAV vector serotypes can be matched to target cell types.
  • the following exemplary cell types can be transduced by the indicated AAV serotypes among others. See Table 1.
  • Tissue/Cell Type Serotype Liver AAV3, AA5, AAV8, AAV9 Skeletal muscle AAV1, AAV7, AAV6, AAV8, Central nervous system AAV1, AAV4, AAV5, AAV8, RPE AAV5, AAV4, AAV2, AAV8, AAV9 Photoreceptor cells AAV5, AA8, AAV9, AAVrh8R Lung AAV9, AAV5 Heart AAV8 Pancreas AAV8 Kidney AAV2, AAV8
  • a pharmaceutical composition including any of the recombinant viral vectors or viral particles described herein.
  • the pharmaceutical compositions can include additional components suitable to, for example, increase delivery (e.g., increase infection of targeted cells and/or increase the range of cells that can be infected), increase stability of the recombinant vector, or decrease immunogenicity of the recombinant vector, for example, an AAV vector.
  • the pharmaceutical compositions can include a pharmaceutically acceptable carrier, excipient, and/or sail.
  • the pharmaceutically acceptable carrier can exclude buffers, compounds, cryopreservation agents, preservatives, or other agents in amounts that can substantially interfere with the delivery or activity of the recombinant AAV vector to a patient.
  • Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the recombinant AAV vector and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • compositions can be delivered to a subject, so as to allow production of an expression product in the cell(s) of the subject.
  • Pharmaceutical compositions include sufficient genetic material that allows the recipient to produce an effective amount of an expression product that modulates FXN expression in a cell and/or treats FA in a subject.
  • the pharmaceutical compositions also contain a pharmaceutically acceptable excipient.
  • excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol.
  • Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • auxiliary substances such as wetting or emulsifying, agents, pH buffering substances, and the like, may be present in such vehicles.
  • auxiliary substances such as wetting or emulsifying, agents, pH buffering substances, and the like.
  • the preparation of pharmaceutically acceptable carriers, excipients and formulations containing these materials is described in, e.g., Remington: The Science and Practice of Pharmacy, 22nd edition, Loyd V. Allen et al. editors, Pharmaceutical Press (2012).
  • compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • genetically modified cells including any of the nucleic acid constructs or recombinant viral vectors described herein.
  • a “genetically modified cell” refers to a cell that has at least one genomic modification as a result of introducing any of the nucleic acid constructs or recombinant viral vectors described herein, into the cell.
  • the genetically modified cells can be in vitro, ex viva or in vivo genetically modified cells.
  • the genetically modified cells can be any suitable genetically modified cell, such as those selected from the group consisting of a human stem cell (for example, a multipotent stem cells, e.g., a mesenchymal stem cell that can differentiate into neurons and cardiomyocytes), human neuron, a human cardiomyocyte, a human smooth muscle myocyte, a human skeletal myocyte, and a human hepatocyte.
  • a human stem cell for example, a multipotent stem cells, e.g., a mesenchymal stem cell that can differentiate into neurons and cardiomyocytes
  • human neuron for example, a multipotent stem cells, e.g., a mesenchymal stem cell that can differentiate into neurons and cardiomyocytes
  • a human cardiomyocyte for example, a multipotent stem cells, e.g., a mesenchymal stem cell that can differentiate into neurons and cardiomyocytes
  • human neuron for example, a multipotent stem cells, e.g.,
  • bone-marrow derived mesenchymal stem cells are isolated from a subject having FA, and genetically modified to insert a nucleic acid construct including a 5′UTR and a nucleic acid sequence encoding human FX.
  • the genetically modified cells are then autologously transplanted back into the subject.
  • the genetically modified cells can be systemically delivered to allow targeted delivery of the grafts to the brain and heart of FA patients. See, for example, Tajiri et al. “Autologous Stem Cell Transplant with Gene Therapy for Friedreich Ataxia” Med. Hypotheses 83(3): 296-298 (2014). Methods for introduction of nucleic acids and vectors for genetic modification of cells are described above.
  • genetic modification refers to any change in the DNA genome (or RNA genome in some cases) of a cell, organism, virus, viral vector, or other biological agent.
  • Non-limiting examples of genetic modifications include an insertion, a deletion, a substitution, a procedure such as a transfection or transformation where exogenous nucleic acid is added to a cell and/or organism, and cloning techniques.
  • Insertions refers to an addition of one or more nucleotides in a nucleic acid sequence. Insertions can range from small insertions of a few nucleotides to insertions of large segments such as a cDNA or a gene.
  • deletion refers to a loss or removal of one or more nucleotides in a nucleic acid sequence or a loss or removal of the function of a gene.
  • a deletion can include, for example, a loss of a few nucleotides, an exon, an intron, a gene segment, or the entire sequence of a gene.
  • deletion of a gene refers to the elimination or reduction of the function or expression of a gene or its gene product. This can result from not only a deletion of sequences within or near the gene, but also other events (e.g., insertion, nonsense mutation) that disrupt the expression of the gene.
  • substitution refers to a replacement of one or more nucleotides in a nucleic acid sequence with an equal number of nucleotides.
  • nucleic acid sequence can result in a “recombinant” sequence.
  • present disclosure provides “recombinant AAV vectors,” which have been genetically modified to include elements disclosed herein.
  • the methods include administering to a subject having FA, a therapeutically effective amount of any of the recombinant AAV particles provided herein.
  • subject an individual.
  • the subject can be an adult subject or a pediatric subject.
  • Pediatric subjects include subjects ranging in age from birth to eighteen years of age.
  • the subject is an animal, for example, a mammal such as a primate, and, more preferably, a human.
  • Non-human primates are subjects as well.
  • the term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.).
  • livestock for example, cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.
  • veterinary uses and medical formulations are contemplated herein.
  • a used throughout “treat,” “treating,” and “treatment” refer to a method of reducing or delaying one or more effects or symptoms of FA.
  • the subject can be diagnosed with FA.
  • Treatment can also refer to a method of reducing the underlying pathology rather than just the symptoms.
  • the effect of the administration to the subject can have the effect of, but is not limited to, reducing one or more symptoms of the disease, a reduction in the severity of the disease, the complete ablation of the disease, or a delay in the onset or worsening of one or more symptoms.
  • a disclosed method is considered to be a treatment if there is about a 10% reduction in one or more symptoms of the disease (e.g., muscle loss, ataxia in anus and legs in a subject, diabetes, cardiomyopathy, etc.) when compared to the subject prior to treatment or when compared to a control subject or control value.
  • the reduction can be about a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between.
  • kits for modulating expression of FXN in a human cell include introducing into the human cell, any of the recombinant AAV vectors provided herein.
  • the cell is in a subject.
  • adenosine triphosphate (ATP) concentration in a human cell of a subject with FA include administering to the subject a therapeutically effective amount of any of the recombinant AAV particles provided herein.
  • the human cell is selected from the group consisting of a neuron, a cardiomyocyte, a smooth muscle myocyte, a skeletal myocyte, and a hepatocyte.
  • the methods include administering a, therapeutically effective amount of any of the recombinant AAV particles provided herein.
  • the human cell is selected from the group consisting of: a neuron, a cardiomyocyte, a smooth muscle myocyte, a skeletal myocyte, and a hepatocyte.
  • an increase can be an increase of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or greater.
  • Increases in the levels of ATP expressed in cells of FA patients can be beneficial for ameliorating one or more symptoms of the disease, increasing long-term survival, and/or reducing side effects associated with other treatments.
  • the recombinant AAV vectors disclosed herein can express increased, yet modulated, levels of FXN, and ATP production by mitochondria can be increased, relative to the disease state.
  • 100,000 to 500,000 cells 500,000 to 1,000,000 cells; 1,000.000 cells to 2,500,000 cells; 2,500,000 to 5,000,000 cells; 5,000,000 to 10,000.000 cells: 10,000,000 to 50,000,000 cells; 50,000,000 to 100,000,000; 100,000,000 to 250,000,000 cells: 150,000,000 to 300,00,000 cells: 250,000,000 to 500,000,000 cells; 500,000,000 to 1,000,000,000 cells: 1,000,000.000 to 5,000,00,000 cells; 5,000,000,000 to 10,000,000,000 cells; 10,000,000,000 to 20,000,00000 cells: 15,000,000,000 to 30,000,000,000 cells; 30,000,000,000 to 50,000,000,000 cells: 50,000,000,000 to 75,000,000,000 cells: or 75,000,000,000 to 100,00000,000 cells in FA patients to whom such recombinant AAV vectors are administered can express increased, yet modulated levels of FXN, and ATP production by mitochondria can be increased, relative to the disease state.
  • effective amount is defined as any amount necessary to produce a desired physiologic response, for example, reducing or delaying one or more effects or symptoms of FA.
  • Effective amounts and schedules for administering the recombinant AAV virions described herein can be determined empirically and making such determinations is within the skill in the art.
  • the dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, unwanted cell death, and the like.
  • the dosage will vary with the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration and severity of the particular condition and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary and can be administered in one or more doses.
  • an effective amount of any of the recombinant AAV virions described herein will vary and can be determined by one of skill in the art through experimentation and/or clinical trials.
  • an effective dose can be from about 10 6 to about 10 15 recombinant rAAV virions, or any values in between this range, for example, about 10 6 , 10 7 , 10 8 , 10 9 . 10 10 , 10 11 , 10 12 , 10 13 10 14 , or 10 15 recombinant AAV particles.
  • the number of rAAV particles administered to a subject may be on the order ranging from about 10 6 to 10 15 vector genomes (vgs)/ml, such as for example, about 10 6 , 10 7 , 10 8 . 10 9 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15 vg/ml.
  • the number of rAAV particles administered to a subject can be from about 10 6 to 10 15 vg/kg, or any values in between these amounts, such as for example, about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 vg/kg.
  • Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.
  • Any of the methods provided herein can further include administering a second therapeutic agent to the subject having FA, for example, a beta blocker, an ACE inhibitor, an antioxidant, a diuretic, an anti-diabetic agent, or a combination thereof.
  • a second therapeutic agent for example, a beta blocker, an ACE inhibitor, an antioxidant, a diuretic, an anti-diabetic agent, or a combination thereof.
  • compositions described herein are administered in a number of ways depending on whether local or systemic treatment is desired.
  • the compositions are administered via any of several routes of administration, intraparenchymal injection, intravenously, intrathecally, intramuscularly, intracistemally, intracoronary injection, intramyocardium injection, intradermally, endomyocardiac injection, or a combination thereof.
  • the compositions are administered canalostomy into the posterior semicircular canal of the subject. Effective doses for any of the administration methods described herein can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the grammatical articles “a”, “an”, and“the”, as used herein, are intended to include “at least one” or “one or more”, unless otherwise indicated, even if “at least one” or “one or more” is expressly used in certain instances.
  • the articles are used herein to refer to one or more than one (i.e., to “at least one”) of the grammatical objects of the article.
  • the use of a singular noun includes the plural, and the use of a plural noun includes the singular, unless the context of the usage requires otherwise.
  • the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. See, In re, Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP ⁇ 2111.03. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “including.”
  • any numerical range recited in this specification describes all sub-ranges of the same numerical precision (i.e., having the same number of specified digits) subsumed within the recited range.
  • a recited range of “1.0 to 10.0” describes all sub-ranges between (and including) the recited minimum value of 1.0 and the recited maximum value of 10.0, such as, for example, “2.4 to 7.6,” even if the range of “ 2 0.4 to 7.6” is not expressly recited in the text of the specification.
  • any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.
  • FXN frataxin
  • compositions and methods for treatment of FA can be treated by modulating expression of the FXN gene via, for example, a viral vector which promotes increased, yet modulated, FXN expression in cells homozygous for GAA trinucleotide repeat alleles.
  • the FXN nucleotide sequence can be operably linked to a 5′ UTR FXN, which can modulate FXN expression.
  • Modulated FXN expression is desired to achieve modulated physiological levels of FXN expression and avoid elevated levels of FXN expression.
  • the non-modulated, elevated physiological levels of FXN can result in reduced mitochondrial respiration, which leads to mitochondrial toxicity.
  • the described compositions and methods represent a novel strategy for treatment of FA, as described and illustrated herein.
  • a first version includes a codon-optimized human FXN nucleotide sequence (SEQ ID NO: I) operably linked to a desmin promoter sequence (SEQ ID NO: 4) and further includes a desmin 5′UTR (SEQ ID NO: 22) operably positioned between the desmin promoter sequence and the human FXN nucleotide sequence.
  • a second version includes the codon-optimized human FXN nucleotide sequence operably linked to the desmin promoter sequence (SEQ ID NO: 4) and further includes a desmin 5′UTR (SEQ ID NO: 22) and 5′UTR FXN sequence (SEQ ID NO: 2) operably positioned between the desmin promoter sequence and the human FXN nucleotide sequence.
  • a third version (SEQ ID NO: 9) is similar to the first version, except that the third version further includes a C-terminal V5 epitope tag in-frame with the human FXN nucleotide sequence.
  • a fourth version (SEQ ID NO: 10) is similar to the second version, except that the fourth version further includes a C-terminal V5 epitope tag in-frame with the human FXN nucleotide sequence.
  • each of the four versions were transfected into separate HEK 293 cell populations, using commercially available transfection reagents according to the manufacturer's instructions. 48 hours post-transfection the four cell populations were collected and total protein was harvested in RIPA buffer. Samples of the total protein (whole cell extract) were subjected to SDS-PAGE and immunoblotting. As indicated in FIG. 4 , blots were probed using commercially available primary antibodies against the V5 epitope ( ⁇ V5), human frataxin ( ⁇ Frataxin), and, as a loading control. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) ( ⁇ GAPDH). Subsequently, blots were probed with HRP-conjugated secondary antibodies.
  • Lane 1 shows results for HEK 293 cells transfected with the first version of the plasmid, including the human FXN nucleotide sequence without a 5′ UTR FXN. No signal was visible on the anti-V5 blot, since no V5 epitope is included in the first plasmid version. Additionally, a signal was visible on the anti-frataxin blot.
  • Lane 2 shows results for HEK 293 cells transfected with the second version of the plasmid, including the human FXN nucleotide sequence with a 5′ UTR FXN. No signal was visible on the anti-V5 blot, since no V5 epitope is included in the second plasmid version.
  • the anti-frataxin signal in lane 2 was less intense than the signal in lane 1.
  • Lane 3 shows results for HEK 293 cells transfected with the third version of the plasmid, including the human FXN nucleotide sequence without a 5′ UTR FXN and with a V5 epitope tag. Signal was visible on the anti-V5 blot, and a signal was visible on the anti-frataxin blot.
  • Lane 4 shows results for HEK 293 cells transfected with the fourth version of the plasmid, including the human FXN nucleotide sequence with a 5′ UTR FXN and with a V5 epitope tag. Signal was visible on the anti-V5 blot, and a signal was visible on the anti-frataxin blot. The anti-frataxin signal and anti-V5 signal in lane 4 were less intense than the corresponding signals in lane 3.
  • Lane 5 shows results for untransfected HEK 293 cells. Signal from endogenous frataxin and GAPDH is visible on the anti-frataxin and anti-GAPDH blots, respectively.
  • Example 1 separate cell populations were transfected with each of the four plasmid versions.
  • SK-N-SH cells a neuroblastoma cell line
  • Transfections and immunoblotting were performed as described in Example 1.
  • lane 1 shows immunoblotting results for untransfected SK-N-SH cells. Signal from endogenous frataxin and GAPDH is visible on the anti-frataxin and anti-GAPDH blots, respectively.
  • Lane 2 shows results for SK-N-SH cells transfected with the second version of the plasmid, including the human FXN nucleotide sequence with a 5′ UTR FXN. No signal was visible on the anti-V5 blot, since no V5 epitope is included in the second plasmid version. Additionally, a signal was visible on the anti-frataxin blot.
  • Lane 3 shows results for SK-N-SH cells transfected with the first version of the plasmid, including the human FXN nucleotide sequence without a 5′ UTR FXN. No signal was visible on the anti-V5 blot, since no V5 epitope is included in the first plasmid version. Additionally, a signal was visible on the anti-frataxin blot. The anti-frataxin signal in lane 2 was less intense than the signal in lane 3.
  • Lane 4 shows results for SK-N-SH cells transfected with the fourth version of the plasmid, including the human FXN nucleotide sequence with a 5′ UTR FXN and with a V5 epitope tag. Signal was visible on the anti-V5 blot, and a signal was visible on the anti-frataxin blot.
  • Lane 5 shows results for SK-N-SH cells transfected with the third version of the plasmid, including the human FXN nucleotide sequence without a 5′ UTR FXN and with a V5 epitope tag. Signal was visible on the anti-V5 blot, and a signal was visible on the anti-frataxin blot. The anti-frataxin signal and anti-V5 signal in lane 4 was less intense than the corresponding signals in lane 5.
  • Example 1 separate cell populations were transfected with each of the four plasmid versions.
  • C2C12 cells a murine myoblast cell line
  • HEK 293 cells of Example 1 Transfections and immunoblotting were performed as described in Example 1.
  • lane 1 shows results for C2C12 cells transfected with the first version of the plasmid, including the human FXN nucleotide sequence without a 5′ UTR FXN. No signal was visible on the anti-V5 blot, since no V5 epitope is included in the first plasmid version. Additionally, a signal was visible on the anti-frataxin blot.
  • Lane 2 shows results for C2C12 cells transfected with the second version of the plasmid, including the human FXN nucleotide sequence with a 5′ UTR FXN. No signal was visible on the anti-V5 blot, since no V5 epitope is included in the second plasmid version. Additionally, a signal was visible on the anti-frataxin blot. The anti-frataxin signal in lane 2 was less intense than the signal in lane 1.
  • Lane 3 shows results for C2C12 cells transfected with the third version of the plasmoid, including the human FXN nucleotide sequence without a 5′ UTR FXN and with a V5 epitope tag. Signal was visible on the anti-V5 blot, and a signal was visible on the anti-frataxin blot.
  • Lane 4 shows results for C2C12 cells transfected with the fourth version of the plasmid, including the human FXN nucleotide sequence with a 5′ UTR FXN and with a V5 epitope tag. Signal was visible on the anti-V5 blot, and a signal was visible on the anti-frataxin blot. The anti-frataxin signal and anti-V5 signal in lane 4 was less intense than the corresponding signals in lane 3.
  • Lane 5 shows results for untransfected C2C12 cells. Signal from endogenous GAPDH is visible on the anti-GAPDH blot.
  • Example 1-3 separate cell populations were transfected with each of the four plasmid versions.
  • C2C12 cells were used, instead of the HEK 293 cells of Example 1.
  • Transfections for the four plasmid versions were performed as described in Example 1. 48 hours post-transfection, the four cell populations were collected and subjected to an adenosine triphosphate (ATP) assay.
  • the ATP assay can measure ATP content in cells and can indicate the relative health of cells' mitochondria. After mitochondrial isolation, mitochondria were assayed using a luciferase assay to quantify the amount of ATP in each sample. Luciferase signal for each sample was analyzed with a standard curve of ATP concentration and measured relative to total protein in each sample.
  • Samples 1-4 are control data for cells transfected with a vector encoding green fluorescent protein (GFP), untransfected cells, and untransfected cells treated with oligomycin A, respectively.
  • GFP green fluorescent protein
  • Oligomycin A is an inhibitor of ATP synthase, and cells treated with oligomycin A serve as a negative control for ATP production.
  • Sample 1 shows results for C2C12 cells transfected with the fourth version of the plasmid, including the human FXN nucleotide sequence with a 5′ UTR FXN and with a V5 epitope tag.
  • the ATP concentration in sample 1 was not statistically different (one-way ANOVNA) from that of the untransfected cells of sample 6.
  • Sample 2 shows results for C2C12 cells transfected with the third version of the plasmid, including the human FXN nucleotide sequence without a 5′ UTR FXN and with a V5 epitope tag.
  • the ATP concentration in sample 1 was increased relative to the ATP concentration in sample 2.
  • Sample 3 shows results for C2C12 cells transfected with the second version of the plasmid, including the human FXN nucleotide sequence with a 5′ UTR FXN.
  • the ATP concentration in sample 3 was not statistically different (one-way ANOVA) from that of the untransfected cells of sample 6.
  • Sample 4 shows results for C2C12 cells transfected with the first version of the plasmid, including the human FXN nucleotide sequence without a 5′ UTR FXN.
  • the ATP concentration in sample 3 was increased relative to the ATP concentration in sample 4.
  • the ATP concentration in sample 4 was decreased relative to that of untransfected control cells of sample 6. *P ⁇ 0.05 (one-way ANOVA).
  • a first plasmid includes a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a chicken beta actin (CBA) promoter sequence (SEQ ID NO: 5) and further includes a CBA 5′UTR (SEQ ID NO: 23) and 5′ UTR FXN sequence (SEQ ID NO: 2) operably positioned between the CBA promoter sequence and the human FXN nucleotide sequence.
  • CBA chicken beta actin
  • a second plasmid (SEQ ID NO: 1 2 ) includes the codon-optimized human FXN nucleotide sequence operably linked to the CBA promoter sequence (SEQ ID NO: 5) and further includes a CBA 5′UTR (SEQ ID NO: 23) operably positioned between the CBA promoter sequence and the human FXN nucleotide sequence.
  • a third plasmid (SEQ ID NO: 13) includes the codon-optimized human FXN nucleotide sequence operably linked to the desmin promoter sequence (SEQ ID NO: 4). The third plasmid further includes the 5′ UTR FXN sequence operably positioned between the desmin promoter sequence and the human FXN nucleotide sequence.
  • a fourth plasmid includes the codon-optimized human FXN nucleotide sequence operably linked to the desmin promoter sequence (SEQ ID NO: 4) and further includes a desmin 5 UTR (SEQ ID NO: 22) and 5′UTR FXN sequence (SEQ ID NO: 2) operably positioned between the desmin promoter sequence and the human FXN nucleotide sequence.
  • a fifth plasmid includes a codon-optimized human FXN nucleotide sequence (SEQ ID NO: 1) operably linked to a desmin promoter sequence (SEQ ID NO: 4) and further includes a desmin 5′UTR (SEQ ID NO: 22) operably positioned between the desmin promoter sequence and the human FXN nucleotide sequence.
  • each of the five versions were transfected into separate C2C12 cell populations, using commercially available transfection reagents according to the manufacturer's instructions.
  • blots were probed using commercially available primary antibodies against either human frataxin or human and mouse frataxin (a. FXN), and, as a loading control, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (a GAPDH). Subsequently, blots were probed with HRP-conjugated secondary antibodies.
  • lane 1 shows results for untransfected C2C12 cells. Signal from endogenous frataxin and GAPDH is visible on the two anti-frataxin blots and the anti-GAPDH blot, respectively.
  • Lane 2 shows results for C2C12 cells transfected with the first plasmid (SEQ ID NO: 11), including the CBA promoter—CBA 5′UTR—5′ UTR FXN—human FXN nucleotide sequence construct. Relative to lane 1, signal in lane 2 was increased.
  • Lane 3 shows results for C2C12 cells transfected with the second plasmid (SEQ ID NO: 12), including the CBA promoter—CBA 5′UTR—human FXN nucleotide sequence construct.
  • Lane 4 shows results for C2C12 cells transfected with the third plasmid (SEQ ID NO: 13), including the desmin promoter—5′ UTR FXN—human FXN nucleotide sequence construct. Relative to lane 1, signal in lane 4 was increased.
  • Lane 5 shows results for C2C12 cells transfected with the fourth plasmid (SEQ ID NO: 8), including the desmin promoter—desmin 5′ UTR—5′ UTR FXN—human FXN nucleotide sequence construct. Relative to lane 4, signal in lane 5 was decreased. Relative to lane 1, signal in lane 5 was of a similar level.
  • Lane 6 shows results for C2C12 cells transfected with the fifth plasmid (SEQ ID NO: 7), including the desmin promoter—desmin 5′ UTR—human FXN nucleotide sequence construct. Signal in lane 6 was increased compared to any one of lanes 1, 4, or 5.
  • RNA samples were also used to extract RNA samples.
  • the RNA samples were then subjected to reverse transcription, and the resulting cDNA samples were then subjected to qPCR.
  • the same five plasmids, as described in Example 5, were used in the present example. Referring to FIG. 9 , relative expression of both beta-actin (Actb) and FXN (Fxn) were determined.
  • Sample 1 which was based on RNA extracted from untransfected cells, showed basal levels of both Actb and FXN expression.
  • Sample 2 was based on RNA extracted from cells transfected with the second plasmid (SEQ ID NO: 12), including the CBA promoter—CBA 5′UTR—human FXN nucleotide sequence construct. Sample 2 showed basal levels of Actb expression and increased levels of FXN expression, relative to the untransfected cells.
  • Sample 3 was based on RNA extracted from cells transfected with the first plasmid (SEQ ID NO: 11), including the CBA promoter—CBA 5′UTR—5′ UTR FXN—human FXN nucleotide sequence construct. Sample 3 showed basal levels of Actb expression and increased levels of FXN expression, relative to the untransfected cells, but decreased levels of FXN expression relative to Sample 2.
  • Sample 4 was based on RNA extracted from cells transfected with the third plasmid (SEQ ID NO: 13), including the desmin promoter—5′ UTR FXN—human FXN nucleotide sequence construct. Sample 4 showed basal levels of Actb expression and increased levels of FXN expression, relative to the untransfected cells.
  • Sample 5 was based on RNA extracted from cells transfected with the fourth plasmid (SEQ ID NO: 8), including the desmin promoter—desmin 5′ UTR—5′ UTR FXN—human FXN nucleotide sequence construct. Sample 5 showed basal levels of Actb expression and basal levels of FXN expression.
  • Sample 6 was based on RNA extracted from cells transfected with the fifth plasmid (SEQ ID NO: 7), including the desmin promoter—desmin 5′ UTR—human FXN nucleotide sequence construct. Sample 6 showed basal levels of Actb expression and increased levels of FXN expression, relative to the untransfected cells, Sample 4, and Sample 5.
  • Sample 6 was based on RNA extracted from cells transfected with the fifth plasmid (SEQ ID NO: 7), including the desmin promoter—desmin 5′ UTR—human FXN nucleotide sequence construct. Sample 6 showed basal levels of Actb expression and increased levels of FXN expression, relative to the untransfected cells, Sample 4, and Sample 5.
  • cassettes containing 1) a tissue-restricted or ubiquitous promoter, and 2) FXN with or without the 5′ untranslated region (UTR) of FXN were designed and tested.
  • the 5′UTR region was selected as a regulatory expression element based on the inherent structures of this region and effects on translation initiation (i.e. post-transcriptional control of gene expression).
  • In vitro experiments demonstrated the effect of transfection-mediated overexpression of frataxin with or without the 5′UTR region of frataxin driven by a modified Desmin (DES) or Chicken Beta Actin (CBA) promoter in a self-complementary terminal repeat (TR) plasmid.
  • DES Desmin
  • CBA Chicken Beta Actin
  • AAV-Des5′ AAV-Des driven 5′UTR-FXN
  • AAV-Des5′ AAV-Des5′
  • AAV-mediated overexpression of 5′UTR-FXN results in toxicity in wild-type mice following intravenous injection or dual-injection routes targeting cerebrospinal fluid (CSF) and skeletal muscle, administered via cisterna magna and intramuscularly (tibialis anterior muscle; TA), respectively.
  • CSF cerebrospinal fluid
  • TA tibialis anterior muscle
  • FXN toxicity used the coding region of the gene without untranslated regions (UTR) that serve as regulatory expression elements to effect translation initiation (i.e. post-transcriptional control of gene expression). Therefore, whether gene expression could be controlled by including the 5′ untranslated region (5′UTR) of FXN was tested.
  • DES modified human desmin
  • CBA chicken ⁇ -actin
  • DES is known for its high transduction in the myocardium, skeletal muscle and CNS, while CBA is a strong ubiquitous promoter leading to high transduction in all cell types. By comparing two different promoters, the aim was to optimize translational efficiency of FXN levels without inducing FXN toxicity.
  • AAV8 triple-capsid mutant AAV87TM (SEQ ID NO:61) was used for delivery of DES-driven 5′UTR-FXN In wild-type mice (AAV8TM-DES-5′UTR-FXN) (plasmid LP1001) (Gilkes et al.
  • Human codon-optimized frataxin (630 bp) 3′UTR or 5′UTR human frataxin (1490 bp and 221 bp, respectively) were cloned in a self-complementary pTR plasmid.
  • the genes of interest (GOI) were driven by a modified human desmin (DES) promoter or chicken ⁇ -actin (CBA) promoter (see Table 2). Plasmid maps are shown in FIGS. 10A-E .
  • the SEQ ID NO. for the nucleic acid sequence of each plasmid is also provided in Table 2.
  • GOIs were synthesized by Integrated DNA Technologies (IDT; Coralville, Iowa, USA) and cloned into pTR-plasmid by restriction enzymes.
  • pLP1001 was generated by cloning “EnhDesPro-intron-5′UTRcoFXNv1” fragment into pds AAV-CBA-EGFP (GenBank: Accession No. MK225672 (SEQ TE NO: 29)) using the restriction enzymes KpnI and SacI.
  • pLP1004 was synthesized by cloning “EnhDesPro-intron-coFXNv1” into pds AAV-CBA-EGFP using restriction site KpnI and SacI.
  • pLP1002 was completed by synthesizing coFXNv1 with Age1 and Sac1 restriction sites which was then cloned into pds-AAV-CBA-EGFP.
  • pLP1003 was completed by synthesizing 5′UTR-intron and cloning it into LP1002 using SalI and SpeI. (Lacerta Therapeutics, Inc. Intellectual Property. Lab notebooks LBN24 LBN25, LBN08)
  • Plasmids were cloned, transformed, and verified by restriction enzyme digest Sma I at Lacerta Therapeutics, Inc. (Alachua, Fla., USA). Following sequence verification (Eurofins), plasmids were sent to Aldevron (Fargo, N. Dak., USA) for large-scale plasmid production.
  • AAV8TM virus expressing DES-5′UTR-FXN was produced in 2 cell stacks (Reference Number 7047 and 7048) by triple transfection in adherent HEK293 cells at the University of Florida, Powell Gene Therapy Center (PGTC), Vector Core Laboratory (Gainesville, Fla., USA).
  • the two cell stacks were pooled, virus was purified by Iodixanol gradient centrifugation followed by an AAVX column and titered by dot blot at PGTC (Table 3).
  • Vector titer was also determined by digital drop PCR (ddPCR) at Lacerta.
  • ddPCR digital drop PCR
  • dot blots reportedly show elevated titers and less accuracy when compared to digital drop PCR. Therefore, in vivo dosing was calculated by the ddPCR titer.
  • AAV8TM-DES-5′UTR-FXN viral titer determined by dot plot (PGTC) or ddPCR (Lacerta) Titer Titer Final Volume Method (vg/ml) (vg/ ⁇ l) (mL) Total vg ddPCR 1.57E+13 1.57E+10 0.86 mL 1.35E+13 Dot Blot 2.54E+13 2.54E+10 1 mL 2.54E+13
  • LTX 401.3 Intravenous Administration of AAV8TM-DES-5′UTR-FXN to Assess Potential Toxicity from FXN Overexpression in Normal Wild-Type Mice
  • Each animal received a single bolus injection of 5E ⁇ 13 vg/kg AAV8TM-DES-5′UTR-FXN in a final volume of 100 ⁇ L through the jugular vein. Vims was prepared by dilution in PBS+0.001% pluronic (excipient) to attain the final dose formulation. An untreated, age matched control animal was also used in this study.
  • LTX 401.4 Dual Administration of AAV8TM-DES-5′UTR-FXN Via Intra Cisterna Magna and Intramuscular Injection to Assess Potential Toxicity from FXN Overexpression in Normal Wild-Type Mice
  • these mice received intramuscular injections (IM) at three different doses (3.70E+8, 8.2E+8, or 1.92E+9 vg/mg tibialis anterior (TA)). Each dose was injected into the right and left TA of one male and one female mouse. To calculate dosage, TA muscle weight was assumed to be 10% of the total body weight.
  • IM intramuscular injections
  • IM injections were prepared to a final volume of 5 ⁇ l by quantity sufficient dilution in excipient.
  • One animal was injected with 10 ⁇ L excipient as a procedural control.
  • an untreated, age matched control animal was included for comparison.
  • AAV8TM-DES-5′UTR-FXN AAV8TM-DES-5′UTR-FXN
  • Table 5 Wild-type C57BL/6J mice (JAX, 000664) were harvested 28-32 days post-injection and tissues were collected as described below, Table 8. Tissues were processed for FXN detection by histology or ELISA to determine biodistribution, human frataxin protein expression, or obvious toxicity following vector administration.
  • Human fibroblast cell lines from Friedreich's Ataxia patients (ID #GM04078 and GM-03816) and healthy donors (ID #GM00969 and GM03651) were obtained from the Corielle Institute (Camden, N.J., USA: Table 7) and cultured in fibroblast growth medium (Promocell C-23010) with 20% fetal bovine serum (Atlanta Biologicals, 511150H). 50 units/ml penicillin, and 50 mg/ml streptomycin (Gibco. 15140-122).
  • Cells were harvested 24 hours after transfection with a cell lifter, then counted and seeded into a 96-well plate at cell density of 5.000 cells/well. The next day, a CyQUANTTM Cell Proliferation Assay (Invitrogen, C7026) was performed according to manufacturer's protocol.
  • Luminescence was measured using a CLARIOstar Microplate Reader (BMG Labtech). A standard curve was generated per the manufacture's protocol and the ATP concentration for each sample was obtained by linear regression analysis. ATP content was normalized to mitochondrial protein concentration. See, Saha et al. “Impact of PYROXD1 deficiency on cellular respiration and correlations with genetic analyses of limb-girdle muscular dystrophy in Saudi Arabia and Sudan,” Physiol Genomics. 2018; 50(11):929-939.
  • Protein concentration was determined by Detergent Compatible (DC) Protein Assay (Bio-Rad).
  • DC Detergent Compatible
  • mitochondrial extract in the amount of 200 ⁇ g total protein was resolved on a 4-12% tricine-polyacrylamide gel (Life Technologies), then transferred onto a nitrocellulose membrane (20 ⁇ m). The membrane was blocked in 5% milk/TBST (0.5% Tween-20, 8 mM Tris-Base, 25 mM Tris-HCl, 154 mM NaCl), then probed with primary mouse anti-frataxin antibody (supernatant) at a 1:1,000 dilution and anti-GAPDH at a 1:1,000 dilution (2118S. Cell Signaling Technologies).
  • DC Detergent Compatible
  • the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies and visualized by chemiluminescence (Millipore) on an iBright CL1000.
  • chemiluminescence Millipore
  • mitochondrial extracts were assayed using Human Frataxin ELISA Kit (ab176112), according to the manufacturer's instructions.
  • Fibroblasts were seeded onto chamber slides (Thermo Scientific, 12-565-8) treated with 10% Matrigel (Corning, CB-40234A) in Dulbecco's Modified Eagle's Medium (Corning #3MT10013CV) after transfection with plasmids, as indicated. At day 4, the growth medium was removed. Cells were washed in PBS, then fixed with 2% Paraformaldehyde in PBS for 10 minutes at room temperature and consecutively washed in PBS for three times. Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes and blocked by the addition of 5% normal goat serum in PBS for 60 minutes. After overnight incubation at 4° C.
  • the titer (vg/mL) for AAV8tm-DES-5′UTR-FXN was determined by dot blot at PGTC and by the QX200 Droplet Digital PCR System from Bio-Rad (QX 200 Droplet Generator and QX200 Droplet Reader Bio-Rad).
  • QX 200 Droplet Generator and QX200 Droplet Reader Bio-Rad QX 200 Droplet Generator and QX200 Droplet Reader Bio-Rad.
  • samples were serially diluted in Nuclease Free Water to 1E3 to 1E2 vg/well to ensure the samples were below the maximum range of analysis (1E4 vg/well). To ensure enough volume for droplet formation, a total volume of 25 uL Mastermix and sample was prepared.
  • the reaction mixture included 1 ⁇ ddPCR Supermix for Probes (No dUTP; Bio-Rad 1863024), 900 nM BGH forward 5′ GCC AGC CAT CTG TTG T 3′ (IDT) (SEQ ID NO: 30) and reverse 5′ GGA GTG GCA CCT TCC A 3′ (IDT) (SEQ ID NO: 31) primers, 250 nM BGH probe 5′ FAM/TCC CCC; GTG/ZEN/CCT TCC TTG ACC/ABkFQ 3′ (IDT) (SEQ ID NO: 32), and 5 ⁇ L of sample diluted in nuclease free water. The mixture was vortexed prior to droplet preparation.
  • Droplets were formed using the QX200 Droplet Generator (Bio-Rad) by adding 20 ⁇ L of the sample mixture into the center wells of a DG8 Cartridge (Bio-Rad, 1864008) followed by 70 ⁇ L of Droplet Generation Oil for Probes (Bio-Rad, 1863005) into the appropriate wells of the cartridge.
  • the cartridge was covered with a DG8 Gasket (Bio-Rad, 1863009) and placed into the Droplet Generator.
  • Newly formed droplets (40 ⁇ L) were carefully pipetted and transferred to a ddPCR 96-well plate (Bio-Rad, 12001925) and covered with a Pierceable Foil Heat Seal (Bio-Rad, 1814040), placed in a PX1 PCR Plate Sealer (Bio-Rad) and heat sealed at 180° C. for 5 seconds.
  • the plate was immediately removed and placed in a C1000 Thermal Cycler (Bio-Rad) at 95° C. for 10 minutes, then 95° C. for 30 seconds, 57.4° C. for 1 minute, and 72° C. for 15 seconds for 42 cycles, followed by 98° C. for 10 minutes and an indefinite hold of 12° C. until the un was stopped.
  • the procedure space was prepared with three designated stations: the animal preparation area, the surgical area, and the recovery/post-op area (Gakuba et al., “General Anesthesia Inhibits the Activity of the “Glymphatic System”,” Theranostics, 8(3), 710-722 (2016)).
  • the Animal Prep Area To reduce the chance of microbial contamination of the sterile surgical field, the animal prep area was positioned on a designated table away from the surgical area. Mice were anesthetized using 2% isoflurane (IL O2) (Falk et al.
  • the surgical area equipment consists of a stainless-steel table, mobile vaporizer anesthesia system, glass bead sterilizer, stereotaxic device, and injection pump; all surfaces were cleaned with 70% alcohol prior to surgery.
  • a sterile drape was placed underneath the stereotax.
  • a heating pad with digital readout was placed on the stereotax where the animal was to be placed and a puppy pad is wrapped around the heating pad once to prevent direct contact of the animal to the heating pad.
  • Two specimen cups, one with chlorhexidine surgical wash, and one with sterile saline rinse were placed on the sterile drape as well as the autoclaved surgical instruments. Surgical instruments were cleaned with soap and water, dried, and sterilized with the glass bead sterilizer in between animals. Autoclaved instruments were used and cleaned a maximum of 10 times before switching to a new set of autoclaved instruments.
  • the animal Upon completion of the surgical procedure, the animal was moved to a clean cage for monitoring and post-op care. This cage was set up so that half of the cage rests on a heating pad with digital readout to minimize hypothermia in the recovering animal; there was a puppy pad between the cage and heating pad to prevent direct contact. The recovery station was close enough to the surgeon/assistant so that recovery could be monitored. Once the animal was able to move normally on its own, showed no sign of distress or pain, and was otherwise bright, alert, and responsive; the animal was be moved back to the cage rack. Animals were monitored daily for the first 5 days post-op, then checked at least every other day until harvest to monitor for complications.
  • LTX-401.3 Intravenous Injection of AAV8TM-DES-5′UTR-FXN into Wild Type Mice
  • mice were anesthetized using vaporized Isoflurane as outlined in the approved IACUC protocol and then weighed (g) in order to calculate analgesia (Rimadyl) administration. Hair was carefully removed from the neck/throat area using depilatory cream (NairTM) and the animal was transferred to the surgical area.
  • the animal was given 1 mL of Lactated Ringers (to replace fluid loss during surgery) and a 10 mg/kg dose of analgesia (Rimadyl) subcutaneously.
  • analgesia Rostyl
  • the animal was placed on the stereotaxic stage in the supine position with its face positioned upwards into the anesthesia face mask.
  • the head was held in an upward position using the anesthetic face mask, the front feet were pulled gently downward and secured in place with tape to expose the neck of the animal and keep contaminated paws out of the surgical area.
  • the flow of vaporized Isoflurane was transferred from the induction chamber at the pre-op station to the anesthesia mask.
  • Anesthetic plane was assessed frequently throughout surgery by observing respirations as well as a toe pedal response, isoflurane levels were adjusted accordingly.
  • the surgical site was aseptically prepared using alternating spiraling outward scrubs of chlorhexidine and 0.9% sterile saline solution beginning at the center of the area from which hair vas removed and working outward towards the periphery, this is repeated at least three tines, or until there is no debris seen on the swab.
  • a 2 cm incision in the skin was made using sterile surgical scissors and forceps to expose the jugular vein.
  • the jugular vein vas located by gently moving away superficial connective and adipose tissue from the incision around the animal's neck. The animal was then ready for injection.
  • a primed and prefilled 29-gauge insulin syringe 100 ⁇ l of diluted virus was inserted to the vein with the bevel of the needle facing upwards.
  • the syringe was aspirated checking for blood flowback into the syringe. If there was no blood drawn up into the syringe, the needle was repositioned and checked again. If there was still no blood return, the needle was removed, and a fresh attempt was made. If excessive bleeding occurred, sterile cotton swabs were used to apply pressure to the vein until bleeding was stopped.
  • the site was sutured, and the surgeon performed the injection on the other side of the neck, this was noted on the surgery record.
  • the syringe was slowly retracted, and pressure was applied to the injection site with a sterile cotton swab to prevent back flow and bleeding; the site vas then cleaned and sutured.
  • Isoflurane delivery was stopped, and the animal was removed from the stereotaxic device. Initial recovery was monitored on the surgical stage before moving the mouse into the recovery cage.
  • LTX-401.4 Intra Cisterna Magna and Intramuscular Injection of AAV8TM-DES-5′UTR-FXN into Wild Type Mice
  • the animal was placed on the stereotaxic device by fixing the head in ear bars and placing the nose in the integrated anesthetic mask.
  • the flow of vaporized Isoflurane was transferred from the induction chamber to the stereotaxic anesthesia mask.
  • Anesthetic plane was assessed frequently throughout surgery by observing respirations as well as a toe pedal response; isoflurane levels were adjusted accordingly.
  • gauze was placed under the heating pad to lift the mouse at an angle so that the spine formed a downward 15° angle with the horizontal line of the ear bars.
  • the anesthetic mask was then adjusted so that the facial surface formed a 15° angle with the vertical line of the stereotaxic arm, this achieves an approximated 90° angle of the head to the spine.
  • the cisterna magna was the highest point of the animal's body and the dura was taut to allow puncture and prevent viral backflow.
  • the surgical site was aseptically prepared using alternating spiraling outward scrubs of chlorhexidine and 0.9% sterile saline solution beginning at the center of the shaved area and working outward towards the periphery, this was repeated at least three times or until there was no debris seen on the swab. After the area was sterilized, a 2 cm incision in the skin was made using sterile surgical scissors and forceps to expose the suboccipital muscles covering the cisterna magna.
  • a 25 ⁇ l Hamilton syringe with a 33-gauge 45°-degree beveled needle attached, pre-filled with 12 ⁇ l (ensuring sufficient volume to deliver 10 ⁇ l) of diluted virus was then placed in the injection pump, mounted on the stereotaxic arm. Subsequently the stereotaxic arm was moved from a 90° vertical angle, down to a 45° angle towards the surgeon. This positioned the needle to be perpendicular to the dura mater. Then the needle of the syringe was positioned using the micromanipulator dials to touch the dura mater (avoiding any blood vessels), the digital readout of the stereotaxic device is then zeroed to mark the start of the dura.
  • the dura mater was pierced.
  • the needle was then retracted back out of the dura using the dials to allow the outflow of cerebrospinal fluid (CSF) to create negative pressure to allow room for the virus.
  • CSF cerebrospinal fluid
  • the outflow of CSF also confirmed that the surgeon was in the correct location.
  • the needle was then reinserted using the dials to position the needle bevel just inside the cisterna magna, approximately 1 mm deep past the recorded dura location. Once the needle was in the correct position, the whole stereotaxic frame wars slowly elevated to form a 30° angle with the table surface to promote the downward flow of virus into the brain.
  • a dollop of sterile Vaseline was placed entirely around the needle at the injection location and on the exposed dura mater to help prevent back flow of vims and CSF.
  • the injection pump set at 1000 nl/min, was then started, and precisely delivered 10 ⁇ l of diluted virus.
  • a tinier was set for one minute to allow for the virus to flow through the subarachnoid space with the CSF to reduce the chance of virus backflow when removing the needle from the cisterna magna.
  • the needle is carefully retracted using the dorsoventral dial. After the needle was retracted, the stereotaxic device was carefully repositioned back to the table level, and the surgical area was cleaned and sutured.
  • anesthetized animals (still on the stereotax) underwent tibialis anterior muscle injections in the left and right leg.
  • the injection site was aseptically prepared using alternating spiraling outward scrubs of chlorhexidine and 0.9% sterile saline solution beginning at the center of the Naired area and working outward towards the periphery, this was repeated at least three times or until there was no debris seen on the swab.
  • Injections were performed into the central portion of the tibialis anterior muscle using a primed 0.5-ml tuberculin syringe with a 29-gauge 45°-degree beveled needle. The needle was inserted into the skin, bevel up, with the needle nearly parallel to the plane of the skin.
  • the viral load was slowly injected. Once the contents of the syringe were fully injected, the needle was slowly retracted to reduce viral backflow. Pressure was applied to the injection site directly after the needle was retracted to help prevent back flow.
  • mice were anesthetized using vaporized Isoflurane as outlined in the approved IACUC protocol and then weighed (g) in order and calculate the amount of analgesia (Rimadyl) needed. Hair was then removed from the back of the head extending from just behind the eyes to the base of the neck using electric clippers. Hair on both the lower hind limbs was then carefully removed using Nair to expose the tibialis anterior muscles. The animal was then transferred to the surgical area.
  • Mitochondrial isolation was performed as described in Preble et al. In summary, after preparation of homogenization buffer, fresh samples were homogenized using the gentleMACSTM Dissociator (Miltenyi Biotec). The homogenate was passed through a 40 ⁇ m filter followed by a 10 ⁇ m filter. The eluate was centrifuged at 9,000 ⁇ g for 10 minutes at 4° C. Pellets were collected and resuspended in ELISA buffer for protein estimation, Immunoblot, and ELISA.
  • Human and mouse frataxin levels were analyzed in isolated mitochondrial fractions from mouse tissues using Human Frataxin ELISA Kit (ab176112) and Mouse Frataxin ELISA Kit (ab199078) according to the manufacturer's instructions.
  • citrate antigen retrieval was performed in a steamer followed by streptavidin/biotin blocking (Vector Laboratories, SP-2002).
  • An anti-mouse IgG (Vector Laboratories, MKB-2213-1) and serum block was also performed before application of the primary mouse anti-frataxin antibody (purified) at 1:300 overnight at 4° C.
  • a biotinylated horse anti-mouse antibody (BA-2000) was applied 1:300 for 10 minutes at room temperature to tissue sections, then washed before application of the fluorescent Dylight 488 conjugated streptavidin (Vector Laboratories, SA-5488-1) at 1:200 for 10 minutes at room temperature.
  • Image acquisition was performed using the Keyence all-in-one microscope (BZ-X810). All H&E slides were scanned at 10 ⁇ magnification with brightfield settings. To image frataxin, heart and quadricep from 401.3 and quadricep and tibialis anterior from 401.4 were scanned at 20 ⁇ . All heart and skeletal muscle sections were scanned using the same settings, heart was scanned at high resolution and muscle with standard resolution. All fluorescent scans included the red channel for contrast and to determine background correction for determination of positive staining.
  • Fibroblasts from healthy individuals (control) and Friedreich's ataxia patients (TA) were treated with plasmid constructs expressing FXN under the control of a CBA or DES promoter with or without a 5′UTR (Table 1).
  • Cells were imaged in a 24-well plate for visualization of cell confluency after transfection with the different constructs ( FIGS. 11A-B ).
  • DNA content was measured by CyQUANT Proliferation Assay ( FIG. 11C ).
  • Untreated cells and cells transfected with a dual reporter plasmid (luciferase-furin2a-tdTomato) under the control of a DES promoter were used as negative and transfection-control, respectively.
  • 11C represents the value at which no toxicity was observed, as determined by the DNA content in normal untreated fibroblasts. All FXN expressing plasmids showed some level of toxicity in both normal and FA fibroblasts. In patient fibroblasts, this level of toxicity remained relatively constant across all FXN plasmid transfections. However, in control fibroblast cell lines, higher DNA content was observed in cells treated with plasmids containing the 5′UTR suggesting this region regulates FXN expression and reduces cellular toxicity.
  • FIGS. 12A-B and ELISA ( FIG. 12C ) assays were conducted on cell lysates of transfected control and diseased fibroblasts to detect human FXN. In both assays, all four FXN expressing plasmids successfully transduced cells. DES-5′UTR-FXN appears to have lower FXN expression compared to DES-FN by Western blot in FA fibroblasts. These results were confirmed and quantified by ELISA showing 60% higher expression in DES-5′UTR-FXN compared to DES-FXN. ELISA showed similar results in control healthy fibroblasts, but the fold difference was negligible in comparison to diseased fibroblasts.
  • Fibroblasts from healthy (control) and FA patients were transfected with 5 pig of plasmid expressing FXN with or without a UTR under the control of CBA promoter (Table 2). Cells that were not transfected (no plasmid) and cells transfected with CBA-GFP were used as negative and transfection control, respectively. Cells were imaged in a 24-well plate for visualization of cell confluency after transfection of constructs ( FIG. 13A ). Cell viability was measured after transfection by CyQUANT assay ( FIG. 13B ). Toxicity analyses revealed CBA-FXN decreased cell viability in control fibroblasts when compared to CBA-5′-FXN. However, FA fibroblasts do not show the same distribution of toxicity ( FIG.
  • FIG. 13C Similarly, ATP content was measured in non- and transfected cells ( FIG. 13D ). Detection of frataxin overexpression by ELISA was ⁇ 16 times higher in CBA-FXN transfected control fibroblasts above endogenous frataxin levels. CBA-5′-FXN and CBA-3′-FXN were ⁇ 10 times higher in expression when compared to endogenous frataxin expression (CBA-GFP). Densitometric analysis was performed after western blot directed against frataxin and GAPDH ( FIG. 13E-13G ). Immunocytochemistry detection of frataxin and tomm20 confirmed co-localization of frataxin in mitochondria ( FIG. 13H ) (19) and staining of control and diseased cells in under each condition was reflective of protein expression ( FIG. 13I ). Titration of plasmid content was performed to reduce toxicity in vitro ( FIGS. 14A-B ).
  • Frataxin levels were measured in the heart, brain, spinal cord, skeletal muscle, liver, and spleen of wild type mice. Normal ranges of mouse frataxin protein was determined after ELISA assay in un-injected animals ( FIG. 15 ). A separate set of wild type mice received an intravenous injection of AAV8TM-DES-5′UTR-FXN at 9 weeks of age to determine potential toxicity resulting from frataxin overexpression in normal animals. Quantification of human frataxin (ELISA) in heart, skeletal muscle, liver, and brain of normal mice following AAV administration results in supra-physiologic levels of FXN expression ( FIG. 16 ). Hematoxylin and eosin staining was conducted to determine if inflammation or toxicity was evident in heart, skeletal muscle, liver, and brain. The staining demonstrated no- to negligible toxicity in the tissues of the injected animals.
  • ELISA human frataxin
  • FIG. 1 ′C Representative images and histochemical analysis of brain regions demonstrate positive detection of frataxin ( FIG. 171 ).
  • the objective of this study was to determine whether a toxicity-dose relationship is observed following dual routes of administration (ICM+IM) of AAV-Des5′.
  • ICM+IM dual routes of administration
  • IM injection TA
  • results of this study support the hypothesis that AAV-Des5′ can express frataxin in targeted tissues without toxicity.
  • the order of the elements in the AAV construct impacts FXN expression.
  • Constructs that do not include a 5′UTR results in highly significant expression (lanes 3 and 6, FIG. 18 ) in C2C12 mouse myoblasts.
  • Inclusion of the 5′ UTR between the intron and FXN results in low FNX expression (lane 5, FIG. 18 ) in C2C12 mouse myoblasts.
  • inclusion of the 5′UTR, an intron and FXN, in that order results in desired FXN expression levels.
  • toxicity was observed in a dose-dependent manner, in normal, control or FA patient fibroblast cell lines, at supraphysiologic FXN expression levels. No toxicity was observed in normal mice following delivery of AAV-5′UTR-FXN in the brain, spinal cord or skeletal muscle. Overexpression of 5′UTR-FXN does not result in obvious toxicity in vivo but loss of cell viability is detected in vitro at highly significant levels of FXN overexpression. Regulation of FXN expression by inclusion of the 5′UTR region reduces the potential for overexpression-induced cellular toxicity.
  • CBA-5UTR-INTRON-FXN the construct containing 5UTR frataxin upstream of sv40 INTRON with CBA promoter
  • CBA FXN the construct containing frataxin with CBA promoter
  • CBA-hFXNpromoter-FXN the construct containing endogenous human frataxin promoter and codon optimized frataxin
  • the cell pellets are collected, and protein isolated by RIPA buffer. A 16% tricine SDS PAGE gel will be run to separate the proteins.
  • the i-blot (Thermofisher Scientific) module will be used to transfer the separated proteins onto a nitrocellulose membrane. The nitrocellulose membrane will be blocked with 5%0 milk in TBST buffer for 2 hours and then probed with primary antibodies and HRP-conjugated secondary antibodies respectively. The western blot is then visualized in i-Bright device after incubation with chemiluminescence solution (Millipore) for 5 minutes.
  • CBA-5UTR-INTRON-FXN the construct containing 5UTR frataxin upstream of sv40 INTRON with CBA promoter
  • CBA FXN the construct containing frataxin with CBA promoter
  • CBA-INTRON-5UTR FXN THE CONSTRUCT containing 5UTR frataxin downstream of sv40 INTRON with CBA promoter
  • CBA-hFXNpromoter-FXN the construct containing endogenous human frataxin promoter and codon optimized frataxin
  • RNA isolation kit Thermofisher Scientific. cDNA will generated from these RNA and qPCR will be conducted to validate the human frataxin copies in each condition.
  • siRNaA will be designed to specifically target the L2 region of the 5′ UTR (SEQUENCE ID 33). C2C12 cells will co-transfected with the plasmids mentioned above and siRNA.
  • the cell pellets will be collected, and proteins isolated by RIPA buffer. A 16% tricine SDS PAGE gel will be run to separate the proteins. After SDS PAGE, an i-blot (Thermofisher Scientific) module will be used to transfer the separated proteins onto a nitrocellulose membrane. The nitrocellulose membrane will be blocked with 5% milk in TBST buffer for 2 hours and then probed with primary antibodies and HRP-conjugated secondary antibodies respectively. The western blot is then visualized in i-Bright device after incubation with chemiluminescence solution (Millipore) for 5 minutes.
  • chemiluminescence solution Millipore
  • the cardiac-specific FXN KO (Fxnflox/mull::MCK-Cre (Jax: 029720)) mouse model has an approximate lifespan ⁇ 9-10 weeks without therapeutic intervention.
  • AAV8TM-CBA-5′-FXN, 5e13 vg/kg virus will be administered intravenously at post-natal day 0 (PND0) or 5 weeks of age; pre-symptomatic and moderate disease stage, respectively.
  • Animals will undergo cardiac MR (11T) to determine cardiac function and morphometry at 9 weeks of age. The goal is to attenuate development of cardiac dysfunction following AAV8TM-CBA-5′-FXN delivery.
  • AAV8TM-CBA-5′-FXN viruses will be made at the Powell Gene Therapy Center, Vector Core at the University of Florida and titered for injections via digital drop PCR.
  • Four week old Fxnflox/null::MCK-Cre mice will be injected with 5 ⁇ 1013 vg/kg dose.
  • Recruitment of animals in each group will follow with a single bolus of test article via intravenous (IV) injection.
  • Body weights will be recorded on a weekly basis. Twenty-eight days post-dose, MRI imaging will be conducted to observe clinically relevant cardiac endpoint in the cardiac mouse model. Left ventricular stroke volume, left ventricular ejection fraction, left ventricular shortening fraction and cardiae output will be measured (Segment software; Medviso).
  • necropsy will include collection of whole blood, brain, spinal cord, dorsal root ganglion, cerebrospinal fluid, heart, left and right quadriceps, left and right tibialis anterior (TA), liver and spleen.
  • Freshly harvested tissues will be subjected to immediate mitochondrial isolation followed by ATP analysis (ATPlite Luminescence Assay. Perkin Elmer). A remaining piece of tissue will be subjected for histological analysis of toxicity, fibrosis, iron deposition and lipid droplets analysis. Mitochondrial will be isolated from frozen tissues for quantitation of human frataxin by ELISA assay (Abcam) and western blot.
  • Fxnflox floxed exon 2 mice
  • Fxnflox mice have a CRISPR/Cas9-generated, Cre-conditional frataxin allele which will be used as a control for the experiment.
  • Mice in groups 1-4 will receive a single bolus of excipient or test article (1.5e11 vg/g brain) via intra-cisterna magna (ICM) injection. Body weights will be recorded on a weekly basis.
  • ICM intra-cisterna magna
  • Behavioral tests using Rotarod, neuroscore, wirehangs and forelimb grip strength tests will be evaluated at 4, 8, 10, 12, 6, 18 and 20 weeks post dose as described in Table 3 and 12, 14, 16, 20 weeks post dose as described (Groups 5-8) [14, 15]. Twenty weeks post-dose, necropsy will include collection of whole blood, brain, spinal cord, dorsal root ganglion, cerebrospinal fluid, heart, left and right quadriceps, left and right tibialis anterior (TA), liver and spleen. Freshly-isolated mitochondria from key tissues will be subjected to ATP analysis.
  • Remaining portions of tissue will be immediately frozen in liquid nitrogen or fixed (4% PFA) for histological analysis (Toxicity-GFAP staining; calbindin staining-rescue of Purkinje neurons and succinate dehydrogenase A-mitochondrial complex II). Frozen tissues will be subjected to mitochondrial isolation and subsequent molecular analysis for quantitation of human FXN by ELISA (Abcam) and western blot. The plan of the study is elaborated in Table 13.

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